U.S. patent number 8,580,549 [Application Number 11/989,927] was granted by the patent office on 2013-11-12 for esterases for separating plastics.
This patent grant is currently assigned to Henkel KGaA. The grantee listed for this patent is Thorsten Eggert, Karl-Erich Jager, Karl-Heinz Maurer, Andreas Michels, Andre Putz. Invention is credited to Thorsten Eggert, Karl-Erich Jager, Karl-Heinz Maurer, Andreas Michels, Andre Putz.
United States Patent |
8,580,549 |
Michels , et al. |
November 12, 2013 |
Esterases for separating plastics
Abstract
The invention relates to agents containing esterases, and to the
use thereof for dressing fibres, in particular, artificial fibres,
washing and cleaning agents comprising esterases and corresponding
washing and cleaning methods, in addition to additional technical
areas of application. The invention also relates to the use of
esterases for protecting against or reducing and/or preventing
pilling, preferably in textiles, particularly plastic fibres, more
preferably polyester fibres, in addition to the use of esterases
for separating the plastics, in particular, polyester compounds.
The invention further relates to novel esterases and to
sufficiently related proteins and to derivatives thereof, agents
containing them and to the use thereof.
Inventors: |
Michels; Andreas (Dusseldorf,
DE), Putz; Andre (Dusseldorf, DE), Maurer;
Karl-Heinz (Erkrath, DE), Eggert; Thorsten
(Essen, DE), Jager; Karl-Erich (Mulheim,
DE) |
Applicant: |
Name |
City |
State |
Country |
Type |
Michels; Andreas
Putz; Andre
Maurer; Karl-Heinz
Eggert; Thorsten
Jager; Karl-Erich |
Dusseldorf
Dusseldorf
Erkrath
Essen
Mulheim |
N/A
N/A
N/A
N/A
N/A |
DE
DE
DE
DE
DE |
|
|
Assignee: |
Henkel KGaA (Duesseldorf,
DE)
|
Family
ID: |
37697171 |
Appl.
No.: |
11/989,927 |
Filed: |
August 3, 2006 |
PCT
Filed: |
August 03, 2006 |
PCT No.: |
PCT/EP2006/007693 |
371(c)(1),(2),(4) Date: |
July 23, 2008 |
PCT
Pub. No.: |
WO2007/017181 |
PCT
Pub. Date: |
February 15, 2007 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20090258406 A1 |
Oct 15, 2009 |
|
Foreign Application Priority Data
|
|
|
|
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Aug 5, 2005 [DE] |
|
|
10 2005 037 659 |
|
Current U.S.
Class: |
435/197; 435/196;
530/350; 435/69.1; 435/19 |
Current CPC
Class: |
C12N
9/18 (20130101) |
Current International
Class: |
C12N
9/18 (20060101); C12N 9/16 (20060101); C12Q
1/44 (20060101); C07K 14/00 (20060101); C12P
21/00 (20060101) |
Field of
Search: |
;435/197,196,69.1,19 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2253063 |
|
May 1973 |
|
DE |
|
0 549 264 |
|
Jun 1993 |
|
EP |
|
0 736 084 |
|
Oct 1996 |
|
EP |
|
09-224664 |
|
Sep 1997 |
|
JP |
|
11-169177 |
|
Jun 1999 |
|
JP |
|
2000-508903 |
|
Jul 2000 |
|
JP |
|
2002-543271 |
|
Dec 2002 |
|
JP |
|
WO-91/02792 |
|
Mar 1991 |
|
WO |
|
WO-97/40144 |
|
Oct 1997 |
|
WO |
|
WO-00/66696 |
|
Nov 2000 |
|
WO |
|
WO-01/81597 |
|
Nov 2001 |
|
WO |
|
WO-2004/016669 |
|
Feb 2004 |
|
WO |
|
Other References
Branden et al., Introduction to Protein Structure, Garland
Publishing Inc., New York, p. 247, 1991. cited by examiner .
Witkowski et al., Biochemistry 38:11643-11650, 1999. cited by
examiner .
Seffernick et al., J. Bacteriol. 183(8):2405-2410, 2001. cited by
examiner .
Zock et al., GenBank accession No. PNBA.sub.--BACSU GI 585706,
2004. cited by examiner .
Zock et al., GenBank accession No. PNBA.sub.--BACSU, GI 68845777
Jul. 5, 2005. cited by examiner .
Veith et al., GenBank AAU39577, Sep. 20, 2004. cited by examiner
.
Evans et al., GenBank accession No. AAG31026, Feb. 17, 2004. cited
by examiner .
Lipman, D. J., et al., "Rapid and sensitive protein similarity
searches", Science, 1985, vol. 227, pp. 1435-1441. cited by
applicant .
Arpigny, J. L., et al., "Bacterial lipolytic enzymes:
classification and properties", Biochem. J., 1999, vol. 343, pp.
177-183. cited by applicant .
Bryan, P. N., "Protein engineering of subtilisin", Biochimica et
Biophysica Acta, 2000, vol. 1543, pp. 203-222. cited by applicant
.
Veith, B., et al., "The complete genome sequence of Bacillus
licheniformis DSM13, an organism with great industrial potential",
J. Mol. Microbiol. Biotechnol., 2004, vol. 7, pp. 204-211. cited by
applicant .
"PnbA (Para-nitrobenzyl esterase)", Database EMBL, XP002432701,
Accession No. Q65MY7, Jan. 25, 2005. cited by applicant .
"PNB esterase 56C8", Database PDB, XP002421545, Accession No. 1C7J,
Feb. 21, 2000. cited by applicant .
"PNB esterase", Database PDB, XP002421546, Accession No. 1QE3, Jul.
12, 1999. cited by applicant .
Spiller, B., et al., "A structural view of evolutionary
divergence", PNAS, 1999, vol. 96, No. 22, pp. 12305-12310. cited by
applicant .
Zock, J., et al., "The Bacillus subtilis pnbA gene encoding
p-nitrobenzyl esterase: cloning, sequence and high-level expression
in Escherichia coli", Gene, 1994, vol. 151, pp. 37-43. cited by
applicant.
|
Primary Examiner: Ramirez; Delia
Attorney, Agent or Firm: Kostiew; Krista A.
Claims
The invention claimed is:
1. A fiber finishing agent comprising an esterase, wherein the
agent prevents or reduces pilling of the fibers or of a fabric into
which the fibers are woven, wherein the esterase is a
para-nitrobenzyl esterase comprising an amino acid sequence that is
greater than 99% identical to the amino acid sequence of SEQ ID
NOs: 1 or 5, and further comprising an ingredient selected from the
group consisting of enzyme stabilizers, surfactants, bleaching
agents, and builders.
2. The agent of claim 1, wherein the fiber finishing is selected
from the group consisting of treatment of textile raw materials and
textile care.
3. The agent of claim 1, wherein the para-nitrobenzyl esterase
comprises an amino acid sequence that is identical to at least one
of SEQ ID NOs: 1 or 5.
4. An isolated para-nitrobenzyl esterase comprising an amino acid
sequence that is greater than 99% identical to the amino acid
sequence of at least one of SEQ ID NOs: 1 or 5.
5. The isolated para-nitrobenzyl esterase of claim 4 comprising an
amino acid sequence that is identical to the amino acid sequence of
at least one of SEQ ID NOs: 1 or 5.
6. A process for the treatment of a textile or a textile raw
material comprising reducing pilling of a fabric by treating the
textile or textile raw material with a para-nitrobenzyl esterase
comprising an amino acid sequence that is greater than 99%
identical to the amino acid sequence of at least one of SEQ ID NOs:
1 or 5.
7. The process of claim 6, wherein the para-nitrobenzyl esterase
comprises an amino acid sequence that is identical to at least one
of SEQ ID NOs: 1 or 5.
8. The process of claim 6, wherein the textile or textile raw
material comprises a synthetic fiber.
9. The process of claim 8, wherein the synthetic fiber comprises
polyester.
10. The fiber finishing agent of claim 1, wherein the surfactants
are selected from the group consisting of non-ionic surfactants,
anionic surfactants and amphoteric surfactants.
Description
RELATED APPLICATIONS
This application is a national stage application under 35 U.S.C.
371 of PCT/EP2006/007693, filed Aug. 3, 2006, which is incorporated
by reference in its entirety and claims benefit of German
application 10 2005 037 659.2, filed Aug. 5, 2005, which is
incorporated by reference in its entirety.
SUBMISSION ON COMPACT DISC
The contents of the following submission on compact discs are
incorporated herein by reference in its entirety: two copies of the
Sequence Listing (COPY 1 and COPY 2) and a computer readable form
copy of the Sequence Listing (CRF COPY), all on compact disc, each
containing: file name: Sequence Listing H06819 13744-53_ST25, date
recorded: Jan. 30, 2008, size: 93 KB.
The present invention relates to agents containing esterases,
especially para-nitrobenzyl esterases, as well as their use for
finishing fibers, in particular, artificial fibres, to detergents
and cleaning agents comprising esterases and corresponding washing
and cleaning methods in addition to further industrial areas of
application. The invention particularly relates to the use of
esterases for protecting against or reducing and/or preventing
pilling, preferably in textiles, particularly artificial fibers,
more preferably polyester fibers, as well as the use of esterases
for the cleaving of plastics, in particular, polyester compounds.
The invention further relates to novel esterases and to
sufficiently related proteins and to derivatives thereof, agents
containing them and to the use thereof.
In general, esterases represent a group of hydrolytic enzymes with
an inherently broad diversity in regard to the substrate and the
reaction type. The substrate specificity and the activation of the
enzymes differ from those of the lipases. It is known that lipases
are activated through the lipid/water interface before they
hydrolyze water-insoluble substrates containing long chain fatty
acid esters. According to Arpigny (Arpigny, Jager, 1999 Biochem. J.
343, pp. 177-183), the esterases (EC 3.1.1) are subdivided into
three different classes: the true lipases (EC 3.1.1.3), carboxyl
esterases (EC 3.1.1.1) and various types of phospholipases.
However, the physiological functions of many esterases, e.g. the
para-nitrobenzyl esterases (PNB esterases), have still not been
elucidated. Normally the esterolytic enzymes are characterized in
that they possess preserved regions that comprise the catalytic
triads as well as in their ability to catalyze a broad spectrum of
reactions. In this respect each hydrolase possesses a specific
stereo preference in regard to a given substrate under specific
reaction conditions, which can be identified as its characteristic
fingerprint. Esterases can be used for the enzymatic hydrolysis of
racemic carboxylic acid esters into their corresponding carboxylic
acids and alcohols. Moreover, they can be used for
transesterification and for the synthesis of esters. The ability of
esterases to be active both in aqueous as well as in non-aqueous
systems makes them important tools for organic synthesis. In this
respect esterases are of particular interest for the synthesis of
enantiomerically pure products.
As a prerequisite for the commercial utilization of esterases it is
desirable to obtain more information on the biocatalytic properties
of the esterases that catalyze reactions of organic compounds.
Pilling is understood to mean fine fibers that are pulled out from
fabrics or knitted fabrics by rubbing and which coil up to form
pills or bobbles that are then only joined to the surface of the
fabric or knitted fabric by a few single fibers. In artificial
fibers the small bobbles of fiber adhere to the surface of the
fabric. Accordingly, solutions are sought after which firstly
reduce the formation of the bobbles (depilling) and secondly
protect the fibers from pilling, such that the unesthetic bobble
formation does not even occur.
The use of cellulases for anti-pilling finishing of cotton and
other natural fibers or fabrics is known. However, the cellulases
are not very suitable for treating the formation of bobbles or
globules in artificial fibers such as for example polyesters or
polyacrylic fibers.
Accordingly the object of the present invention was to discover new
suitable ways to counteract pilling, especially of polyester or
polyacrylic fibers and/or to get rid of or disintegrate the
resulting bobble-like structures. A further object consisted in the
provision of novel esterases, especially for use in detergents
and/or cleaning agents.
These objects are achieved by the use of esterases for finishing
fibers, especially artificial fibers, in particular for
anti-pilling finishing, detergents and cleaning agents as well as
finishing agents for fabrics, in particular fabric post treatment
and pre-treatment agents that comprise esterases, corresponding
finishing, washing and cleaning processes and the use of esterases
in detergents and cleaning agents as well as additional industrial
applications. The invention particularly relates to the use of
esterases for protecting against or reducing or preventing pilling,
preferably in textiles, particularly artificial fibers, more
preferably polyester fibers, as well as the use of esterases for
the cleaving of plastics, in particular, polyester compounds.
When pilling is prevented, or particularly with textiles, the
bobbles on the fibers are reduced, then the garment has a higher
wearing comfort, in particular due to the improved softness, and
the garment retains a good or new appearance much longer.
A further subject of the invention relates to novel esterases and
to sufficiently related proteins and to derivatives thereof, agents
containing them and to the use thereof in particular for the
finishing of fibers.
Additional subsidiary objects consisted in the provision of nucleic
acids that code for these types of esterases, and the provision of
vectors, host cells and manufacturing processes that can be
utilized for the production of such esterases. In addition, it was
the intention to provide suitable agents, especially detergents and
cleaning agents, suitable washing and cleaning processes as well as
suitable end-use applications for these types of esterases.
The object is achieved by the use of esterases (EC 3.1.1),
especially carboxyl esterases (EC 3.1.1.1), preferably
para-nitrobenzyl esterases, especially those that can be obtained
from microorganisms, particularly bacteria, preferably from
bacteria of the Bacillus species.
Those esterases, which according to the methods 2.4 cited in the
examples exhibit a specific activity towards the substrate
bis-(p-methylbenzoic acid) ester of ethylene glycol of 0.1 to 30,
preferably 0.6 to 20, particularly 0.7 to 15, quite particularly
preferably 0.9 to 10, even more strongly preferably 1 to 5,
particularly 1.1 to 4, quite particularly preferably 1.5 to 3
(.mu.mol liberated acid)/(min mg enzyme), are particularly suitable
for use in the inventive agents as well as also for the inventive
use, listed below, of such esterases. These esterases have proved
to be particularly advantageous for use in the inventive agents or
uses and processes.
Esterases are particularly suitable that contain amino acid
sequences that are identical to the amino acid sequences listed in
SEQ ID NO. 1, 2, 4, 6, 11-26 to at least 50%, preferably 60%,
particularly 70%, preferably at least to 80%, particularly
preferably at least to 90%, preferably at least to 95% and quite
particularly preferably to 100%, or are homologous to at least 90%,
preferably at least 95% and quite particularly preferably to
100%.
Further solutions to the object or to the subsidiary objects and
therefore to each of the individual subjects of the invention
consist in nucleic acids whose sequences are sufficiently similar
to the nucleotide sequences given in SEQ ID NO. 3, 4, 7 or 8 or
which code for inventive esterases, in corresponding vectors,
cells, or host cells and manufacturing processes. In addition,
suitable agents, especially detergents and cleaning agents,
suitable washing and cleaning processes as well as suitable end-use
applications for these types of esterases are provided. Finally,
industrial applications for the discovered esterases are
defined.
Esterases that are particularly preferred for use in the inventive
agents and also for the inventive uses listed further below are
those that are homologous to the protein sequence listed in Seq. ID
Nr. 12 to at least 50%, at least 55%, particularly at least 60%,
preferably at least 65%, particularly preferably at least 70%,
advantageously at least 75%, quite particularly preferably at least
80%, particularly preferably at least 85%, at least 90%, at least
95%, at least 99%, particularly 100%.
In the context of the present application, a protein is understood
to mean a polymer composed of natural amino acids, which is
essentially linear in structure and which assumes in the main a
three dimensional structure for carrying out its function. In the
present application, the 19 naturally occurring L-amino acids that
serve as building blocks of proteins, are designated with the
customary international 1- and 3 letter codes. The combination of
one of these designations with a number designates which amino acid
group is in which position in the relevant protein. Analogous
designations are established for point mutations. Positional data
refer, when not otherwise stated, to each of the mature forms of
the protein in question, i.e. without the signal peptide (see
below).
In the context of the present application, an enzyme is understood
to mean a protein that has a specific biochemical function.
Numerous proteins are so called preproteins, i.e. formed together
with a signal peptide. Included among these is the N-terminal part
of the protein, whose function is mainly to guarantee the expulsion
of the formed protein from the production cells into the periplasma
or the surrounding medium and/or its correct folding. Subsequently,
the signal peptide is split off from the remaining protein under
natural conditions by a signal peptidase, such that this exercises
its original catalytic activity without the first present
N-terminal amino acids.
Due to their enzymatic activity, the mature peptides, i.e. the
enzymes processed after their production, are preferred over the
preproteins for industrial applications.
In the context of the present application, nucleic acids are
understood to mean the molecules that are naturally constructed
from nucleotides, which serve as information carriers and code for
the linear amino acid sequence in proteins or enzymes. They can be
present as a single strand, as a complementary single strand to
this single strand or as a double strand. Nucleic acid DNA is
preferred as the naturally, long lasting information carrier for
molecular biological work. On the other hand, an RNA is formed for
the realization of the invention in natural surroundings, such as
for example in an expression cell, which is why RNA molecules that
are essential for the invention also represent embodiments of the
present invention. (c-) DNA molecules can once again be derived
from them, for example by reverse transcription.
The information unit of a nucleic acid corresponding to a protein
is also designated as a gene in the context of the present
application. In DNA the sequences of both complementary strands
have to be taken into account in each of all three possible reading
frames. In addition, it has to be taken into account that different
codon triplets can code for the same amino acids, with the result
that a specific amino acid sequence can be derived from a plurality
of different and nucleotide sequences exhibiting possibly only
slight identity (degeneracy of the genetic code). Moreover, various
organisms exhibit differences in the use of these codons. On these
grounds, both amino acid sequences as well as nucleotide sequences
have to be included in considerations of the field of protection,
and listed nucleotide sequences are only to be regarded as an
example of coding for a specific amino acid sequence.
Using today's generally known methods, such as for example chemical
synthesis or the polymerase chain reaction (PCR) in combination
with molecular biological and/or protein chemical standard methods,
it is possible for the person skilled in the art to manufacture,
with the help of known DNA sequences and/or amino acid sequences,
the complete genes. Such methods are known to the person skilled in
the art. In particular, this is possible if one can revert to a
strain deposited in a collection of strains. For example, with PCR
primers, which can be synthesized by means of a known sequence,
and/or through isolated mRNA molecules, the gene in question can be
synthesized from such strains, cloned and optionally further
treated, for example mutagenized.
Modifications of the nucleotide sequence, as can be brought about
by known molecular biological methods, are called mutations. Known
types depend on the nature of the modification, for example
deletion mutations, insertion mutations or substitution mutations
or those in which various genes or parts of genes are fused
together (shuffling); they are gene mutations. The associated
organisms are called mutants. The proteins derived from mutated
nucleic acids are called variants. Thus, for example deletion-,
insertion-, substitution mutations or fusions lead to deletion-,
insertion-, substitution mutants or fusion genes and at the protein
level to corresponding deletion-, insertion- or substitution
variants or fusion proteins.
In the context of the present invention, vectors are understood to
mean elements that consist of nucleic acids, which comprise a gene
of interest as the characterizing nucleic acid region. They are
able to establish the gene as an independent replicating, stable
genetic element in a species or a cell line over several
generations or cell divisions. Vectors, particularly when used in
bacteria, especially plasmids, are therefore circular genetic
elements. In gene technology, a differentiation is made, on the one
hand, between those vectors that serve the storage and thereby to a
certain extent also the technical genetic work, the so called
cloning vectors, and on the other hand, those that fulfill the
function of realizing the gene of interest in the host cells, i.e.
to enable the expression of the protein in question. These vectors
are called expression vectors.
Both bacteria cells and eukaryotic cells, which comprise the cited
vectors, are generally called cells regardless of their
differences. Such cells that comprise a vector, especially an
expression vector and thus can be stimulated to express a
transgene, are called host cells because they harbor the relevant
genetic system.
Homologization is the comparison of a nucleic acid- or amino acid
sequence with that of known genes or proteins. It is conducted, for
example, over an alignment. The measure of homology is a percentage
rate of the identity, as can be determined for example according to
the methods given by D. J. Lipman and W. R. Pearson in Science 227
(1985), pp. 1435-1441. This result can refer to the whole protein
or to each of the attributable regions. A further broad homology
term, the similarity, also factors into the evaluation conserved
variations, i.e. amino acids with similar chemical activity,
because these execute mostly similar chemical activities inside the
protein. For nucleic acids, only the percentage rate of identity is
known.
By means of homologization, the functions of individual sequence
regions as well as the enzymatic activity of the whole enzyme under
consideration can be deduced from the amino acid- or nucleotide
sequence. Homologous regions of different proteins are those with
comparable functions, which can be recognized by identity or
conserved exchanges in the primary amino acid sequence. They
include single amino acids, the smallest regions, so called boxes
that are only a few amino acids long, up to long regions in the
primary amino acid sequence. Functions of the homologous regions
are thus understood to also mean the smallest partial functions of
the function exercised by the whole protein, such as for example
the formation of single hydrogen bonds for complexing a substrate
or transition complex. Other regions of the protein, which are not
involved in the actual enzymatic reaction, can qualitatively or
quantitatively modify it. This can concern, for example, the enzyme
stability, the activity, the reaction conditions or the substrate
specificity.
The term esterase is understood to mean an enzyme with esterase
activity or that of an esterase, moreover all functions are
therefore understood going from the functions of the few amino acid
groups of the catalytically active center, as they result from the
action of the total remaining protein or a part or several parts of
the remaining protein on the actual catalytically active regions.
In the context of the invention, such modifying functions on their
own or partial activities, in so far as they support an esterase
reaction, are also regarded as esterolytic activity. Such auxiliary
functions or partial activities include, for example, the binding
of a substrate, of an intermediate or end product, the activation
or the inhibition or intervention of a regulating influence on the
hydrolytic activity. This can also concern, for example, the
formation of a structural element that lies far removed from the
active center. The second prerequisite for a protein to be
considered according to the invention as having esterase activity,
is however, that there results a hydrolysis of ester bonds by the
chemical behavior of the actual active groups alone or additionally
by the action of the modifying part. Moreover, it is also possible
that the activities of other esterases can also be qualitatively or
quantitatively modified through one or more parts of the inventive
protein, for example. This influence from other factors is also
regarded as esterase activity. Active enzymes are also those
esterases, whose activity at a given point in time is blocked for
example by an inhibitor. What is crucial is their ability in
principle in regard to the corresponding esterase reaction.
Preferred enzymes, in particular para-nitrobenzyl esterases, in the
context of the invention are particularly those enzymes that are
capable of catalysing the hydrolysis of para-nitrophenyl acetate.
In addition, all enzymes characterized in data banks as
para-nitrobenzyl esterases are preferred and are understood to be
among the inventive para-nitrobenzyl esterases.
Those esterases are particularly preferred, which according to the
methods 2.4 cited in the examples exhibit a specific activity
towards the substrate bis-(p-methylbenzoic acid) ester of ethylene
glycol of 0.1 to 30, preferably 0.6 to 20, particularly 0.7 to 15,
quite particularly preferably 0.9 to 10, even more strongly
preferably 1 to 5, particularly 1.1 to 4, quite particularly
preferably 1.5 to 3 (.mu.mol liberated acid)/(min*mg enzyme). These
esterases have proved to be particularly advantageous for use in
the inventive agents or uses and processes.
Those esterases are particularly preferred which are homologous to
the protein sequences listed under Seq. ID Nr. 12 to at least 50%,
at least 55%, particularly at least 60%, preferably at least 65%,
particularly preferably at least 70%, advantageously at least 75%,
quite particularly preferably at least 80%, particularly preferably
at least 85%, at least 90%, at least 95%, at least 99%. The
esterase according to Seq ID Nr. 12 or fragments of this esterase,
in particular those fragments that exhibit an esterase activity,
are quite particularly preferred. These esterases particularly
exhibit a surprisingly good stability towards higher temperatures
and higher pH. It was determined that these esterases are stable
for long periods or active at alkaline pH and also at a temperature
of greater than or equal to 60.degree. C.
Consequently, the inventive esterases are suitable for use
particularly at alkaline pH at high temperatures, in particular in
heavy-duty detergents that usually exhibit an alkaline pH, and
particularly for hot washes (washing temperature at 95.degree.
C.).
Fragments are understood to mean all proteins or peptides, which
are smaller than natural proteins or those that correspond to
completely translated genes, and for example can also be obtained
synthetically. Due to their amino acid sequences, they can be
assigned to the relevant complete proteins. For example, they can
assume the same structure or exercise proteolytic activities or
partial activities. Fragments and deletion variants of starting
proteins are in principle very similar; while fragments depict
rather smaller debris, the deletion mutants rather lack only short
regions, and therefore only a few partial functions.
In the context of the present application, chimeric or hybrid
proteins are understood to mean those proteins that are composed of
elements that originate naturally from different polypeptide chains
from the same organism or from different organisms. This procedure
is also called shuffling or fusion mutagenesis. The sense of such a
fusion consists in, for example, providing or modifying an
enzymatic function with the help of the fused-on inventive protein
part.
"Proteins obtained by means of insertion mutation" are understood
to mean those variants that have been obtained by known methods of
inserting a nucleic acid fragment or protein fragment into the
starting sequences. Due to their fundamental similarity, they are
classified as chimeric proteins. They differ from those only in the
proportion of the size of the unchanged part of the protein to the
size of the whole protein. In these insertion mutated proteins, the
share of foreign protein is less than in chimeric proteins.
Inversion mutagenesis, meaning a partial reversal of the sequence,
can be regarded as a special form of both deletion as well as of
insertion. The same is true for new groupings of different
molecular parts that differ from the original amino acid sequence.
It can be regarded both as a deletion variant, as an insertion
variant as well as a shuffling variant of the original protein.
In the context of the present application, derivatives are
understood to mean proteins, whose particular amino acid chain has
been chemically modified. Such derivatizations can be effected
biologically, for example, by the host organism in connection with
the protein biosynthesis. Molecular biological methods for example
can be employed for this, for example cotransformation with genes
that provide the modification in question. However, derivatizations
can also be effected chemically, for example by the chemical
transformation of a side chain of an amino acid or by the covalent
bonding of another compound onto the protein. This type of compound
can also concern other proteins for example that are bonded to the
inventive protein through a bifunctional chemical compound, for
example. These types of modification influence, for example, the
substrate specificity or the binding strength to the substrate or
provide a temporary blocking of the enzymatic activity in the case
where the attached substance is an inhibitor. This can be
meaningful for the storage period, for example. Similarly,
derivatization is also understood to mean the covalent bonding to a
macromolecular support.
In the context of the present invention, all enzymes, proteins,
fragments, fusion proteins and derivatives, in so far as they do
not need to be explicitly treated as such, are assimilated under
the generic term proteins.
"Activity of an enzyme" is understood as its efficiency in each
technical field in question, preferably in the context of a
suitably targeted agent. This is based on the actual enzymatic
activity, but in addition is dependent on further relevant factors
for the process in question. These include for example stability,
substrate binding, interaction with the material supporting the
substrate or interactions with other ingredients, especially
synergists.
In the context of the present application, "washing performance" or
"cleaning performance" of a detergent or cleaning agent is
understood to mean the effect that the agent in question produces
on the soiled article, for example fabrics or objects with hard
surfaces. Individual components of such agents, for example
individual enzymes are assessed in regard to their contribution to
the washing performance or cleaning performance of the total
detergent or cleaning agent. This is because the enzymatic
properties of an enzyme do not allow a straightforward analysis of
its contribution to the washing performance of an agent. Other
factors play a role, such as stability, substrate binding, binding
onto the goods being cleaned or interactions with other ingredients
of the detergent or cleaning agent, especially synergies during
removal of the soils.
The present invention is based on the finding that esterases,
especially selected from Lipase P and p-nitrobenzyl esterases,
preferably such enzymes that occur naturally in bacteria, quite
particularly in bacteria of the genus Bacillus, particularly
preferably from the Bacillus licheniformis and subtilis species,
are suitable for reducing or for preventing pilling on textile
fibers and textile fabrics.
Polyesters are polymers whose basic components are held together
through ester bonds. The "homopolymers" can be classified into two
groups according to their chemical structure, the hydroxycarboxylic
types (AB-polyesters) and the dihydroxydicarboxylic acid types
(AA-BB-polyesters). The former are manufactured from only a single
monomer by e.g. polycondensation of an .omega.-hydroxycarboxylic
acid or by ring opening polymerization of cyclic esters (lactones).
The synthesis of the latter, on the other hand, is effected by
polycondensation of two complementary monomers, e.g. a diol and a
dicarboxylic acid. Branched and crosslinked polyesters are obtained
by the polycondensation of tri- or polyhydric alcohols with
polyfunctional carboxylic acids. The polycarbonates (polyesters of
carbonic acid) are also generally counted among the polyesters.
AB-Type-P. (I) are inter alia polyglycolic acids (polyglycolides,
R.dbd.CH.sub.2), polylactic acids (polylactides,
R.dbd.CH--CH.sub.3), poly(.beta.-hydroxybutyric acid)
[poly(3-hydroxybutyric acid), R.dbd.CH(CH.sub.3)--CH.sub.2],
poly(.epsilon.-caprolactone)s [R.dbd.(CH.sub.2).sub.5] and
polyhydroxybenzoic acids (R.dbd.C.sub.6H.sub.4).
Pure aliphatic AA-BB-type polyesters (II) are polycondensates of
aliphatic diols and dicarboxylic acids, which inter alia are used
as products with terminal hydroxyl groups (as polydiols) for the
manufacture of polyester polyurethanes [e.g. polytetramethylene
adipate, R.sup.1.dbd.R.sup.2.dbd.(CH.sub.2).sub.4] AA-BB-type
polyesters with the greatest industrial volumes are those from
aliphatic diols and aromatic dicarboxylic acids, particularly the
polyalkylene terephthalates [R.sup.2.dbd.C.sub.6H.sub.4, with
polyethylene terephthalate (PET) R.sup.1.dbd.(CH.sub.2).sub.2,
polybutylene terephthalate (PBT) R.sup.1.dbd.(CH.sub.2).sub.4 and
poly(1,4-cyclohexane dimethylene terephthalate)s (PCDT)
R.sup.1.dbd.CH.sub.2--C.sub.6H.sub.10--CH.sub.2] as the most
important representatives. The properties of these types of
polyesters can be broadly varied and matched to various
applications by the additional use of other aromatic dicarboxylic
acids (e.g. isophthalic acid) or by adding diol mixtures during the
polycondensation.
Purely aromatic polyesters are the polyacrylates, which include
inter alia the poly(4-hydroxybenzoic acid) (Formula I,
R.dbd.C.sub.6H.sub.4), polycondensates of Bisphenol A and phthalic
acid (Formula II,
R.sup.1.dbd.C.sub.6H.sub.4C(CH.sub.3).sub.2C.sub.6H.sub.4,
R.sup.2.dbd.C.sub.6H.sub.4) or also those of bis-phenols and
phosgene.
In addition to the previously cited saturated polyesters,
unsaturated polyesters can also be manufactured from unsaturated
dicarboxylic acids. These polyester resins have acquired industrial
significance, in particular as unsaturated polyester resins
(UP-resins).
Polyesters are also found in nature, where they are formed from
hydroxycarboxylic acids (e.g. depsides and depsipeptides).
Poly(.beta.-hydroxybutyric acid) serves as a storage substance for
many bacteria. Sand bees or Miner bees produce polyester from
18-hydroxyoctadecanoic acid and 20-hydroxyeicosanoic acid to line
their nests.
According to a preferred embodiment, in particular esterases
selected from Lipase P and para-nitrobenzyl esterases are employed
as the esterases. These PNB-esterases are characterized in that
they provide a particularly good protection of textile fibers
against pilling.
In the context of the invention, suitable para-nitrobenzyl
esterases (p-nitrobenzyl esterases, pNB-esterases, EST-B) are
especially those enzymes as described in the patent applications
U.S. Pat. Nos. 5,468,632, 5,906,930, 5,945,325, EP 0 549 264, which
are incorporated herein by reference in their entirety. The
p-nitrobenzyl esterases disclosed therein are all inventively
preferred.
Furthermore, those para-nitrobenzyl esterases are particularly
preferred with an amino acid sequence which is identical to the
amino acid sequences listed under Seq. ID Nr. 1, 2, 4 6 or 11-25 to
at least 50%, preferably to at least 60%, particularly to at least
70%, to at least 80%, to at least 85%, to at least 86%, to at least
87%, to at least 88%, to at least 89%, to at least 90%, to at least
91%, to at least 92%, to at least 93%, to at least 94%, to at least
95%, to at least 96%, to at least 97%, to at least 98%, to at least
99%, or 100% and/or are homologous to at least 80%, to at least
85%, to at least 86%, to at least 87%, to at least 88% %, to at
least 89%, to at least 90%, to at least 91%, to at least 92%, to at
least 93%, to at least 94%, to at least 95%, to at least 96%, to at
least 97%, to at least 98%, to at least 99% or 100%.
Those para-nitrobenzyl esterases are particularly preferred which
are 95%, particularly preferably 98%, especially 100% identical
with the listed amino acid sequences (1, 2, 4, 6, 11-25).
Those esterases are particularly preferred, which according to the
methods 2.4 cited in the examples exhibit a specific activity
towards the substrate bis-(p-methylbenzoic acid) ester of ethylene
glycol of 0.1 to 30, preferably 0.6 to 20, particularly 0.7 to 15,
quite particularly preferably 0.9 to 10, even more strongly
preferably 1 to 5, particularly 1.1 to 4, quite particularly
preferably 1.5 to 3 (.mu.mol liberated acid)/(min mg enzyme). These
esterases have proved to be particularly advantageous for use in
the inventive agents or uses and processes.
Those esterases are particularly preferred which are homologous to
the protein sequences listed under Seq. ID Nr. 12 to at least 50%,
at least 55%, particularly at least 60%, preferably at least 65%,
particularly preferably at least 70%, advantageously at least 75%,
quite particularly preferably at least 80%, particularly preferably
at least 85%, at least 90%, at least 95%, at least 99%, especially
100%.
A further subject of the invention is the use of esterases,
particularly esterases from Lipase P and p-nitrobenzyl esterases,
for cleaving polyalkylene terephthalates, particularly polyethylene
terephthalates (abb.: PET or PETE).
Likewise the esterases can be employed to cleave or degrade
plastics, particularly polyesters and/or plasticizers in plastics.
Phthalates are frequently used in plastics as plasticizers in order
to improve the properties of the plastics during processing or
use.
The invention relates to the partial or total degradation of molded
objects, fabrics, coatings, adhesive bonds or foams made of
enzymatically biologically degradable polymers. It particularly
relates to the enzymatic degradation of polyesters. The polymer
degradation process can be carried out in a variety of ways:
The polymer is added to the aqueous enzyme-containing solution. The
biologically degradable polymer can be added as a film, sheet or
pellet. Molded articles can be added whole or in small pieces.
Coated or bonded materials or materials that were coated with
biologically degradable polymers or produced with adhesives, such
as for example paper or cardboard as well as coated paper or coated
cardboard, can be added as is or in small pieces to the
enzyme-containing solution.
In addition, the aqueous enzyme-containing solution can be coated
or sprayed onto the coating or the molded article to be
degraded.
The described process of enzymatic degradation of biological and
enzymatically degradable polymers and blends produced thereof can
be inventively employed for example to include chemicals, active
substances, auxiliaries, enzymes, microorganisms, plant seeds in
(e.g. capsules and microcapsules) and their targeted release by the
addition of enzymes.
Thus, by the use of the inventive process, e.g. in garbage
treatment installations, the environment can be more quickly rid of
biologically and/or degradable polymers or their mixtures.
Those esterases are particularly preferred, which according to the
methods 2.4 cited in the examples exhibit a specific activity
towards the substrate bis-(p-methylbenzoic acid) ester of ethylene
glycol of 0.1 to 30, preferably 0.6 to 20, particularly 0.7 to 15,
quite particularly preferably 0.9 to 10, even more strongly
preferably 1 to 5, particularly 1.1 to 4, quite particularly
preferably 1.5 to 3 (.mu.mol liberated acid)/(min mg enzyme). These
esterases have proved to be particularly advantageous for use in
the inventive agents or uses and processes.
Those esterases are particularly preferred which are homologous to
the protein sequences listed under Seq. ID Nr. 12 to at least 50%,
at least 55%, particularly at least 60%, preferably at least 65%,
particularly preferably at least 70%, advantageously at least 75%,
quite particularly preferably at least 80%, particularly preferably
at least 85%, at least 90%, at least 95%, at least 99%, especially
100%. Likewise, mutations of the enzyme listed under SEQ ID NO. 12
that exhibit a further improved inventive action are particularly
preferred.
A further subject of the invention is inventively employable
naturally formed esterases that are obtainable from supernatant
cultures or after cell digestion.
The nucleotide sequence of the inventive novel esterases from
Bacillus subtilis (17A1) is listed in the sequence protocol of the
present application under SEQ ID NO. 3. It comprises 1470 bp. The
derived amino acid sequence is listed in SEQ ID NO. 1. It includes
489 amino acids followed by a stop codon.
The nucleotide sequences of the inventive novel esterases from
Bacillus licheniformis (19C5) are listed in the sequence protocol
of the present application under SEQ ID NO. 4. They each comprise
1470 bp. The derived amino acid sequence is listed in SEQ ID NO. 2.
It includes 489 amino acids followed by a stop codon.
Because of the recognizable agreements and the connections to the
other cited esterases, the esterases are to be regarded as
p-nitrobenzyl esterases.
Consequently, a subject of the present invention is any esterase
with an amino acid sequence that is identical to at least 70% to
the amino acid sequences listed in SEQ ID NO. 1, 2, 5 or 6.
Among those that are increasingly preferred are those whose amino
acid sequence is at least 72%, 74%, 76%, 78%, 80%, 82%, 84%, 85%,
86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%
identical to the amino acid sequences listed in SEQ ID NO. 1, 2, 5
or 6, preferably 1 or 2. It is expected that their properties are
increasingly similar to those found.
An embodiment of this inventive subject matter is each esterase
that is derived from a nucleotide sequence that is at least 70%
identical (assuming an identical codon usage) to one of the
nucleotide sequences listed in SEQ ID NO. 3, 4, 7 or 8.
Among those that are increasingly preferred are those which derive
from a nucleotide sequence that is at least 72%, 74%, 76%, 78%,
80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% and 100% identical to the nucleotide sequence listed
in SEQ ID NO. 3.
Among those that are also increasingly preferred are those which
derive from a nucleotide sequence that is at least 72%, 74%, 76%,
78%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% and 100% identical to the nucleotide sequence
listed in SEQ ID NO. 4.
It is therefore to be expected that these nucleic acids code for
proteins whose properties are increasingly similar to those of the
inventive esterases.
The most preferred embodiment of this inventive subject matter is
each esterase whose amino acid sequence is altogether identical
with the amino acid sequences listed in SEQ ID NO. 1, 2, 5 or 6
and/or whose amino acid sequence is altogether identical with one
of the amino acid sequences derived from the nucleotide sequences
listed in SEQ ID NO. 3, 4, 7 or 8.
The esterases that are newly discovered and provided by the present
application from Bacillus subtilis or licheniformis are those of
this type.
They are esterases that are not known in the prior art. They can be
isolated, manufactured and applicable, as listed in the examples.
They are further characterized in that when used in an appropriate
agent, their activity approximates or even exceeds that of the
enzymes established for this purpose.
For the development of industrial esterases that in particular are
applicable in detergents, as a natural microbially formed enzyme it
can serve as a starting point to be optimized for the desired
application by means of mutagenetic methods that are known per se,
for example point mutagenesis, fragmentation, deletion, insertion
or fusion with other proteins or protein fragments or by other
modifications. These types of optimizations can be for example
adaptations to the effects of temperature, pH fluctuations, redox
conditions and/or other influences that are relevant to the
industrial field of use. An improved oxidation resistance,
stability against denaturing agents or proteolytic degradation,
against high temperatures, acidic or strongly alkaline conditions,
a reduction in immunogenicity or allergenic activity for example
are desired.
The mutagenesis processes involve one of the associated nucleotide
sequences that are listed in SEQ ID NO. 3, 4, 7 or 8 or the
sufficiently similar nucleotide sequences that are illustrated
below as a separate inventive subject matter. Suitable molecular
biological methods are described in the prior art, for example in
pertinent handbooks such as that by Fritsch, Sambrook und Maniatis
"Molecular cloning: a laboratory manual", Cold Spring Harbour
Laboratory Press, New York, 1989.
Further embodiments of the present invention are all proteins or
fragments derived from one of the above described, inventive
esterases by fragmentation or deletion mutagenesis, with increasing
preference for at least 25, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 343 and
360 amino acids already located in the starting molecule connected
at the beginning, internally or at the end of the starting amino
acid sequence.
Here, it is increasingly preferred that each of those proteins or
fragments derived by fragmentation or deletion mutagenesis are
identical to the sequences listed in SEQ ID NO. 1, 2, 7 or 8 to at
least 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
and to 100%.
"Inventive fragments" are understood to mean all proteins or
peptides, which are smaller than those homologous proteins that
correspond to those of SEQ ID NO. 1 or SEQ ID NO. 2, but which
match them in the corresponding partial sequences. These fragments
can be, for example single domains or fractions that do not match
with the domains. Such fragments may be cheaper to manufacture, no
longer possess certain possibly detrimental characteristics of the
starting molecule, such as an activity-reducing regulation
mechanism, or develop a more favorable activity profile. These
types of protein fragments may also be manufactured by
non-biosynthetic methods, for example chemically. The chemical
synthesis is for example advantageous when chemical modifications
have to be carried out subsequent to the synthesis.
The proteins that are also obtainable by deletion mutation are to
be assigned to the fragments due to their fundamental similarity.
Deletion mutagenesis is particularly helpful for removing
inhibiting regions. Both a specialization as well as an enlargement
of the application range of the protein can result from the
deletions.
Proteins and signal peptides obtained from pre-proteins by cleavage
of the N-terminal amino acids can also be considered as naturally
formed fragments or deletion-mutated proteins. This type of
cleavage mechanism can be used for example in order to provide
specific cleavage points in recombinant proteins with the aid of
certain sequence regions that are identified by signal peptidases.
Thus activation and/or deactivation in vitro of inventive proteins
can be effected. As an example may be cited the signal peptide
according to Seq ID No 9 (MMRKKSFWLGMLTAFMLVFTMAFSDSASA).
Further embodiments of the present invention are all from an
inventive esterase described above or from a corresponding fragment
by insertion mutagenesis, by substitution mutagenesis and/or by
fusion with at least one other protein or proteins derived from
protein fragments.
Inventive chimeric proteins possess in the broadest sense an
esterolytic activity. This can be performed or modified by a part
of a molecule that derives from an inventive protein. The chimeric
proteins can lie across their whole length as well as outside the
above claimed region. The sense of such a fusion consists in, for
example, providing or modifying a certain function or partial
function with the help of the fused-on inventive protein part. It
is irrelevant in the context of the present invention whether such
a chimeric protein consists of a single polypeptide chain or a
plurality of sub-units. The latter alternative can be effected for
example posttranslationally or first after a purification step by
means of a targeted proteolytic cleavage by breaking down a single
chimeric polypeptide chain into several.
Therefore it is possible, for example to equip an inventive protein
or fragment thereof through peptidic linkers or directly as the
fusion protein with binding domains from other proteins, for
example the cellulose binding domains, and thereby to more
effectively design the hydrolysis of the substrate. Such a binding
domain could also derive from an esterase, for example to
strengthen the binding of the inventive protein to an esterase
substrate. This increases the local esterase concentration and can
be advantageous in specific applications, for example in the
treatment of raw materials. Similarly, inventive proteins can also
be linked for example with amylases or cellulases so as to execute
a dual function.
The inventive proteins that can be obtained by insertion mutation
are assigned to the inventive chimeric proteins due to their
fundamental similarity. Substitution variations also belong here,
i.e. those in which single regions of the molecule have been
substituted with elements from other proteins.
The significance of insertion and substitution mutagenesis is as in
hybrid formation, to combine individual properties, functions or
partial functions of inventive proteins with those of other
proteins. This also includes a shuffling or novel combination of
partial sequences from various esterases over to obtained variants.
In this way proteins can be obtained that beforehand had not yet
been described. Such techniques enable drastic effects down to very
subtle modulations in activity.
Preferably such mutations are carried out according to a
statistical process, known to the person skilled in the art,
classified as regional directed evolution, such as for example
according to the StEP method, the random priming recombination, the
DNA shuffling or recursive sequence recombination or the RACHITT
method. Such processes are necessarily coupled with a selection or
screening process subsequent to the mutagenesis and expression, so
as to recognize variants having the desired properties. As these
techniques apply to the DNA level, the starting point for the
biotechnological production is made available with each of the
associated newly produced genes.
Inversion mutagenesis, meaning a partial reversal of the sequence,
can be regarded as a special form of both deletion as well as of
insertion. Such variants can likewise be targeted or randomly
produced.
Active molecules are preferred over inactives, as for example in
particular the exercised esterolysis is important in the
application fields listed below.
The above listed fragments also possess, in the broadest sense, an
esterolytic activity, for example for complexing a substrate or for
forming a structural element required for hydrolysis. They are
preferred when they can be already considered for use for the
hydrolysis of an ester bond, without the need for the presence of
further esterase components. This relates to the activity that an
esterase per se can execute; the possible simultaneously required
presence of buffer substances etc. remains unaffected.
An interaction of different molecular parts for the hydrolysis
naturally exists in deletion mutants rather than in fragments and
ensues in particular in fusion proteins, quite particularly those
that emanate from a shuffling of related proteins. Thus, in so far
as an esterolytic function in the broadest sense is sustained,
modified, specified or also first achieved, then the deletion
variants and the fusion proteins are inventive proteins. Preferred
representatives of this inventive subject are among those that are
capable of hydrolyzing a substrate without the need for the
presence of further esterase components.
A preferred embodiment is illustrated by all those proteins,
protein fragments or fusion proteins that have been listed, which
are characterized in that they are further derivatised.
Derivatives are understood to mean those proteins that are derived
from the listed proteins by an additional modification. These types
of modifications can influence for example the stability, substrate
specificity or the binding strength to the substrate or the
enzymatic activity. They can also serve to reduce the allergenicity
and/or immunogenicity of the protein and thereby increase its skin
compatibility, for example.
Such derivatizations can be effected biologically, for example by
the produced host organism in connection with the protein
biosynthesis. Here, couplings of low molecular weight compounds
such as lipids or oligosaccharides are particularly emphasized.
However, derivatizations can also be effected chemically, for
example by the chemical transformation of a side chain or by the
covalent bonding of another, for example macromolecular compound
onto the protein. For example, the coupling of amines on carboxylic
groups of an enzyme is possible in order to change the isoelectric
point. Macromolecules, such as proteins, for example can be bonded
through e.g. bifunctional chemical compounds to inventive proteins.
Thus it is possible for example to provide an inventive protein
over a linker with a specific binding domain. These types of
derivatives are particularly suitable for use in detergents or
cleaning agents. In analogy with WO 00/01831, esterase inhibitors
can also be bonded through linkers, especially amino acid linkers,
to the inventive proteins. Couplings with other macromolecular
compounds, such as polyethylene glycol, improve the molecule in
regard to further properties, such as stability or skin
compatibility.
In the broadest sense, derivatives of inventive proteins can also
be understood to mean preparations of these enzymes. Depending on
extraction, work up or preparation a protein can be blended with
various other materials, for example from the cultures produced by
microorganisms. Certain other materials can also be purposely added
to a protein, for example to increase its storage stability.
Therefore, all preparations of an inventive protein are also in
accordance with the invention. This is also independent of whether
this enzymatic activity is actually displayed by a specific
preparation. It may be desired that it possesses no or only limited
activity during storage, and first develops its esterolytic
function at the time of use. This can be controlled for example by
suitable accompanying substances.
A preferred embodiment is illustrated by all those proteins,
protein fragments or fusion proteins that have been listed so far
and which are characterized in that they are additionally
stabilized.
In this way their stability during storage and/or during their use,
for example during the washing process, is increased such that
their activity lasts longer and is consequently boosted. Coupling
to polymers, for example, can increase the stability of inventive
esterases. This requires that prior to use in suitable agents, the
proteins be bonded with such polymers by means of a coupling
step.
Stabilizations that are possible by point mutagenesis of the
molecule itself are preferred. No further process steps would then
be required after having extracted the protein. Some point
mutations that are suitable for this are known from the prior
art.
Other possibilities are for example: the exchange of proline for
certain amino acid groups; the introduction of polar or charged
groups on the surface of the molecule;
Another possibility for stabilizing against increased temperature
and the effect of surfactants can reside in stabilization by the
exchange of amino acids in close proximity to the N-terminus with
those that come into contact with the remainder of the molecule
through non-covalent interactions and consequently contribute to
maintaining the globular structure.
Preferred embodiments are those in which the molecule is stabilized
by a plurality of methods. It can be assumed that several
stabilizing mutations act additively.
A preferred embodiment is represented by all proteins, protein
fragments, fusion proteins or derivatives, which are characterized
in that they have at least one antigenic determinant in common with
one of the above described inventive proteins, protein fragments,
fusion proteins or derivatives.
The secondary structural elements of a protein and its three
dimensional folding are decisive for the enzymatic activities.
Thus, domains that significantly differ from each other in their
primary structure can form spatially largely conformable structures
and therefore make possible the same enzymatic behavior. Such
commonalities in the secondary structure are usually identified as
autologous antigenic determinants of antiserums or of pure or
monoclonal antibodies. Similar proteins or derivatives can be
detected and classified in this way by means of immunochemical
cross reactions. Consequently such proteins that may possibly not
be classified by their degree of homology in the primary structure
but arguably by their immunochemical affinity to the above defined
inventive proteins, protein fragments, fusion proteins or
derivatives are also precisely included in the scope of protection
of the present invention.
A preferred embodiment is illustrated by all those proteins,
protein fragments, fusion proteins or derivatives that have been
listed up to now, which are characterized in that they are obtained
from a natural source, in particular from a microorganism.
For example they can be single cell fungi or bacteria. Mostly they
can be more easily extracted and handled than the multicellular
organisms or the cell cultures derived from metazoa; although these
can represent reasonable options for specific embodiments and are
thus not fundamentally excluded from the subject of the
invention.
It is possible that naturally occurring products can indeed
manufacture an inventive enzyme; however under the investigated
conditions this only expresses to a limited extent and/or releases
into the surrounding medium. However, this does not rule out
suitable environmental conditions or other factors from being
experimentally determined and that their application could
stimulate a commercially reasonable production of the inventive
protein. Such a regulation mechanism can be purposely employed for
biotechnological production. If this is also not possible then they
can still be used for isolating the associated gene.
Among these, those from gram-positive bacteria are particularly
preferred. This is because they do not possess an external membrane
and thus immediately release secreted proteins into the surrounding
medium.
Those from gram-positive bacteria of the genus Bacillus are quite
particularly preferred.
A priori, bacillus esterases possess favorable characteristics for
various fields of application. They include a certain stability
towards increased temperature, oxidizing or denaturing agents. In
addition, most experience has been obtained with microbial enzymes
in regard to their biotechnological production, for example
concerning the construction of cost-effective cloning vectors, the
selection of host cells and growth conditions or the estimation of
risk, such as for example the allergenicity. Furthermore, bacilli
are established as production organisms having a particularly high
production performance in industrial processes. The wealth of
experience acquired for the manufacture and use of these enzymes is
of great benefit to the inventive further development of these
enzymes. This concerns for example their compatibility with other
chemical compounds, such as, for example, the ingredients of
detergents or cleaning agents.
Among those of the Bacillus species, once again those from the
species Bacillus subtilis or licheniformis are preferred.
The embodiments of the inventive enzymes were originally obtained
from these. The associated sequences are given in the sequence
transcript. The above described variants can be manufactured from
them or related strains by the use of standard microbiological
methods, such as, for example PCR and/or the known point
mutagenesis methods.
The nucleic acids that serve to accomplish the invention represent
a further solution to the problem of the invention and thereby a
separate subject matter of the invention.
Nucleic acids form the starting point for virtually all molecular
biological investigations and developments as well as the
production of proteins. This includes in particular the gene
sequencing and the deduction of the associated sequence of amino
acids, each type of mutagenesis (see above) and the protein
expression.
Mutagenesis for the development of proteins having defined
characteristics is also called "protein engineering". Examples of
characteristics that are optimized have already been described
above. Such a mutagenesis can be targeted or carried out with
random methods, for example with a screening and selection method
directed to the final activity of the cloned genes, for example by
hybridization with nucleic acid sensors, or on the gene products,
the proteins, for example regarding their activity. Further
development of the inventive esterases can be organized according
to the considerations presented in the publication "Protein
engineering" by P. N. Bryan (2000) in Biochim. Biophys. Acta,
Volume 1543, pp. 203-222.
Consequently, a subject of the present invention is any nucleic
acid coding for an esterase, whose nucleotide sequence is identical
to at least 70% to the nucleotide sequences listed in SEQ ID NO. 3,
4, 7 or 8 (for comparable codon usage).
Among those that are increasingly preferred are those whose
nucleotide sequence is at least 72%, 74%, 76%, 78%, 80%, 82%, 84%,
85%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and
100% identical to the nucleotide sequences listed in SEQ ID NO. 3,
4, 7 or 8.
It is therefore to be expected that these nucleic acids code for
proteins whose properties are increasingly similar to those of the
esterases from Bacillus.
Additional representatives of this subject matter of the invention
are all nucleic acids that code for one of the above described,
inventive proteins, protein fragments, fusion products or
derivatives.
The nucleic acids that code for the above described, preferred
forms are correspondingly preferred, in particular also the nucleic
acids obtained by mutagenesis.
The nucleic acids that code for protein fragments are especially
explicitly included in the scope of protection of the present
invention. For such oligonucleotides, all three reading frames have
to be taken into account in both the sense as well as in the
anti-sense direction. They can be used, particularly in the
polymerase chain reaction (PCR), as the starting point for the
synthesis of related nucleic acids, for example for the
amplification of related genes from natural organisms. They can
also serve for the production of chimerics by a PCR-based shuffling
process. Other shuffling processes, such as for example the
recombining ligation reaction (RLR) also involve oligonucleotides
that correspond to randomly or targeted selected protein fragments.
Anti-sense oligonucleotides can also be employed for expression
regulation, for example.
In accordance with the abovementioned statements, the following are
increasingly preferred among the above described inventive nucleic
acids: those that are obtained from a natural source, in particular
from a microorganism; among the above, those wherein the
microorganism concerns a gram-positive bacterium; among the above,
those wherein the gram-positive bacterium concerns one of the genus
Bacillus; and; among the above, those wherein the genus Bacillus
concerns a Bacillus subtilis or licheniformis.
A separate subject matter of the invention is formed by vectors
that comprise one of the previously identified, inventive nucleic
acid regions, especially one that codes for one of the previously
identified proteins, protein fragments, fusion proteins or
derivatives.
In order to deal with the relevant inventive nucleic acids, and
therefore in particular to prepare the production of inventive
proteins, said acids are suitably ligated in vectors. Such vectors
and the associated working methods are extensively described in the
prior art. A great number and a broad variation of vectors are
commercially available, both for cloning as well as for expression.
These include for example vectors that are derived from bacterial
plasmids, bacteriophages or viruses, or predominantly synthetic
vectors. Furthermore, they are differentiated according to the
nature of the cell types, in which they are capable of establishing
themselves, for example according to vectors for gram-negative, for
gram-positive bacteria, for yeasts or for higher eukaryotes. The
form suitable starting points for molecular biological and
biochemical investigations, for example, as well as for the
expression of the gene in question or associated proteins.
In one embodiment the inventive vectors concern cloning
vectors.
In addition to the storage, the biological amplification or the
selection of the gene of interest the cloning vectors are suitable
for its molecular biological characterization. At the same time
they represent transportable and storable forms of the claimed
nucleic acids and are also starting points for molecular biological
techniques that are not linked with cells, such as for example PCR
or in vitro mutagenesis processes.
Preferably, the inventive vectors are expression vectors.
Such expression vectors are the basis for the realization of the
corresponding nucleic acids in biological production systems and
hence for the production of the associated proteins. Preferred
embodiments of this subject matter of the invention are expression
vectors that carry genetic elements required for expression, for
example the natural localizing promoter originating before the gene
or a promoter from another organism. These elements can be arranged
in the form of a so called expression cassette, for example.
Alternatively, individual or all regulation elements can also be
prepared from the relevant host cell. The expression vectors are
particularly preferably matched in regard to further
characteristics, such as, for example the optimum copy number, the
chosen expression system, especially the host cells (see
below).
In addition, it is advantageous for a high expression rate if the
expression vector comprises preferably only the gene in question as
the insert and no larger 5'- or 3'-non coding regions. Such inserts
are obtained for example if the fragment obtained after statistical
treatment of the chromosomal DNA of the starting strain with a
restriction enzyme after the sequencing has been purposely cut once
more before the integration into the expression vector.
A separate subject matter of the invention is formed by cells that
comprise one of the previously identified, inventive nucleic acid
regions, especially one that codes for one of the previously
identified inventive proteins, protein fragments, fusion proteins
or derivatives, preferably on one of the previously identified,
inventive vectors.
These cells comprise the genetic information for the synthesis of
an inventive protein. They enable for example the amplification of
the corresponding gene, but also its mutagenesis or transcription
and translation and finally the biotechnological production of the
protein in question. This genetic information can be integrated
either extrachromosomally as the single genetic element, i.e. for
bacteria present in the plasmidic localization or be integrated
into a chromosome. The choice of a suitable system depends on the
issues, such as for example the nature and period of storage of the
gene, or of the organism or the nature of the mutagenesis or
selection. Thus, in the prior art for example are described
mutagenetic and selection methods based on bacteriophages--and
their specific host cells--for the development of detergent
enzymes.
Preferably this concerns host cells that express one of the
previously described, inventive protein, protein fragments, fusion
proteins or derivatives or can be stimulated to their expression,
in particular by employing one of the previously identified,
inventive nucleic acid regions, quite particularly by employing one
of the previously identified expression vectors.
The host cells that form the proteins enable their biotechnological
production. For this they must have received the gene in question,
suitably with one of the previously described vectors and whose
transcription must be capable of translation and preferably the
possible additional modification steps.
In principle, all organisms, i.e. prokaryotes, eukaryotes or
cyanophytae are suitable host cells for protein expression. Those
host cells are preferred, which can be genetically handled with
ease, for example in relation to the transformation with the
expression factor and its stable establishment and the regulation
of the expression, for example single cell fungi or bacteria. In
addition, preferred host cells are those with a good
microbiological and biotechnological handleability. For example
this relates to ease of cultivation, high growth rates, low demands
on fermentation media and good production rates and secretion rates
for foreign proteins. Laboratory strains that are geared to
expression are preferably selected. They are commercially available
or can be obtained from generally accessible collections of
strains. Theoretically, each inventive protein can be obtained in
this way from a plurality of host organisms. The optimum expression
system for the individual case must be experimentally determined
from the abundance of different systems available from the prior
art.
Host cells that are themselves esterase-negative are particularly
advantageous.
Preferred embodiments are illustrated by such host cells that, due
to suitable genetic elements, can be regulated in their activity,
for example by the controlled addition of chemical compounds, by
changing the conditions of cultivation or as a function of the
respective cell density. This controllable expression enables a
very cost effective production of the proteins of interest.
Suitably the gene, expression vector and host cell are matched to
one another, for example in regard to the genetic elements required
for expression (ribosome binding points, promoters, terminators) or
the codon usage. The codon usage for example can be optimized in
that in the gene of those codons that are only poorly translated
from the host in question, each of the same meaning are replaced by
those that are more useful for the respective host.
Preferred among these are host cells that are characterized in that
they are bacteria, in particular those that secrete the formed
protein into the surrounding medium.
Bacteria are characterized by short generation times and low
demands on the cultivation conditions. In this manner, cost
effective processes can be established. Moreover, there exists an
extensive wealth of experience with bacteria in fermentation
technology. For a specific production in particular cases requiring
factors such as nutrient sources, rates of product formation, time
requirements etc., to be experimentally determined, the most varied
gram-negative or gram positive bacteria can be suitable.
With gram-negative bacteria, such as E. coli, a plurality of
proteins is secreted into the periplasmic space. This can be
advantageous for specific applications. In contrast, gram-positive
bacteria, such as for example Bacilli, immediately release secreted
proteins into the nutrient medium surrounding the cells, from which
medium, according to another preferred embodiment, the expressed
inventive proteins can be directly purified.
A process is disclosed in the application WO 01/81597, wherein it
is claimed that gram-negative bacteria also discharge the expressed
proteins. Such a system is also suitable for manufacturing
inventive proteins. Consequently, those of the species Escherichia
coli or Klebsiella are preferred as host cells, particularly those
of the strains E. coli JM 109, E. coli DH 100B, E. coli DH 12S or
Klebsiella planticola (Rf). In order for the produced proteins to
be released, suitable microbiological modifications and/or suitable
vectors described in this application are required.
As host cells, preferred bacteria are those that are characterized
in that they are gram-positive bacteria, in particular that they
belong to the genus Bacillus, quite particularly to the species
Bacillus lentus, Bacillus licheniformis, Bacillus
amyloliquefaciens, Bacillus subtilis or Bacillus alcalophilus.
An embodiment of the present invention utilizes B. licheniformis or
B. subtilis itself to (homologously) express inventive proteins. In
contrast, however, the heterologous expression is preferred. For
this, bacteria of the genus Bacillus are preferred because on
production grounds they are the best characterized among the
gram-positive bacteria. These particularly include those of the
species B. licheniformis, B. amyloliquefaciens, B. subtilis or
other species or strains of B. alcalophilus. With these species
there exists pertinent experience concerning the enzyme
manufacture. These related species additionally dispose of a
similar codon usage, such that their protein synthesis apparatus is
by nature suitably attuned.
A further advantage is that with this process a mixture of
inventive proteins can be obtained with the enzymes that are
endogenously formed from the host strains. This type of
coexpression also emanates from the application WO 91/02792. When
this is not required, the esterase genes that are naturally present
in the host cell have to be permanently or temporarily inactivated
(see above).
Further preferred are host cells that are characterized in that
they are eukaryotic cells, in particular those that
post-translationally modify the formed protein.
Examples of suitable eukaryots are fungi like actinomycetes or
yeasts like saccharomyces or kluyveromyces, as well as thermophilic
fungal expression systems. These are particularly suitable for the
expression of temperature stable variants. Modifications that
eukaryotic systems carry out, particularly in connection with the
protein synthesis, include for example the binding of low molecular
weight compounds such as membrane anchors or oligosaccharides.
These types of oligosaccharide modifications can be desirable for
lowering the allergenicity. A coexpression with the enzymes that
are naturally formed from these types of cells, such as for example
cellulases, can also be advantageous.
Processes for manufacturing an inventive protein represent an
independent subject matter of the invention.
Accordingly, each process is claimed for manufacturing one of the
above described, inventive protein, protein fragment, fusion
protein or derivative by employing one of the above described,
inventive nucleic acids and/or by employing one of the above
described, inventive vectors and/or by employing one of the above
described, inventive cells.
They include, for example, chemical synthesis processes that are
particularly economically expedient for shorter fragments.
In contrast, however, all molecular biological, microbiological or
biotechnological manufacturing processes that are established in
the prior art and already discussed in detail above are preferred.
Therefore for example, with the previously identified DNA- and
amino acid sequences, such as, for example those that can be
derived from the sequence transcript, preferably with those from
SEQ ID NO. 1, 2, 5 and 6 themselves, corresponding oligonucleotides
and oligopeptides up to the complete genes and proteins can be
synthesized using known molecular biological methods.
Starting from the known esterase producing microorganisms, for
example based on the example in the present application, one can
identify and isolate additional natural producers of esterases,
determine their esterase-gene- and/or amino acid sequences and
correspondingly further develop the specifications made here. Such
bacterial species can also be cultivated and employed for
appropriate manufacturing processes. Analogously, new expression
vectors can be developed. Embodiments of the present invention
based on the associated nucleic acid sequences can also be
cell-free expression systems, in which the protein biosynthesis is
reconstructed in vitro. All of the elements listed above can also
be combined in new processes to manufacture the inventively
employable proteins. A plurality of possible combinations of
process steps is conceivable for each inventive protein, such that
optimum processes have to be experimentally determined for each
practical single case.
A separate subject matter of the invention is represented by agents
comprising one of the previously described, inventive proteins,
protein fragments, fusion proteins or derivatives.
All types of agents, in particular mixtures, formulations,
solutions etc., whose suitability is improved by the addition of
one of the inventive proteins described above, are hereby included
in the scope of protection of the present invention. Depending on
the field of application, this can concern for example solid
mixtures, for example powders with freeze dried or encapsulated
proteins, or agents in gel or liquid form. Preferred compositions
comprise for example buffer substances, stabilizers and/or reaction
partners of the esterases and/or other ingredients that are
synergistic with the esterases. Among these in particular are
agents for the application areas listed further below. Additional
application areas emerge from the prior art and are illustrated for
example in the handbook "Industrial enyzmes and their applications"
by H. Uhlig, Wiley-Verlag, New York, 1998.
Detergents or cleaning agents comprising one of the previously
described, inventive proteins, protein fragments, fusion proteins
or derivatives make up the preferred embodiment of the subject
matter of this invention.
As is shown in the embodiments of the present application, it was
surprisingly determined that the particularly preferred esterases
from Bacillus, i.e. already the wild type enzyme stands out, in
that when used in a suitable detergent or cleaning agent, it at
least approximates or partially even surpasses the contributions to
washing or cleaning performance of enzymes established for this
purpose.
This embodiment of the invention also includes all the possible
types of cleaning compositions--both concentrates and compositions
to be used without dilution--for use on a commercial scale, in
washing machines or in hand washing or cleaning. These include, for
example, detergents for fabrics, carpets or natural fibers, for
which the term "detergent" is used in the present invention. These
also include, for example, dishwashing detergents for dishwashing
machines or manual dishwashing detergents or cleaners for hard
surfaces, such as metal, glass, china, ceramic, tiles, stone,
painted surfaces, plastics, wood or leather, for which the term
"cleaning agent" is used in the present invention. Any type of
detergent or cleaning agent represents an embodiment of the present
invention, providing it is enriched by an inventive protein,
protein fragment, fusion protein or derivative.
Embodiments of the present invention include all types established
by the prior art and/or all required usage forms of the inventive
detergents or cleaning agents. These include for example solid,
powdered, liquid, gel or pasty agents, optionally from a plurality
of phases, compressed or non-compressed; further included are for
example: extrudates, granulates, tablets or pouches, both in bulk
and also packed in portions.
In a preferred embodiment, the inventive detergent or cleaning
agents comprise the above described, inventive or inventively
useable proteins, protein fragments, fusion proteins or derivatives
in an amount of 0.0001 .mu.g to 480 mg, preferably 0.005 .mu.g to
420 mg, particularly preferably 0.02 .mu.g to 360 mg, quite
particularly preferably 0.05 .mu.g to 240 mg per gram of the
agent.
In addition to an inventive protein, protein fragment, fusion
protein or derivative, an inventive detergent or cleaning agent
optionally comprises further ingredients such as enzyme
stabilizers, surfactants, e.g. non-ionic, anionic and/or amphoteric
surfactants, and/or bleaching agents, and/or builders, as well as
optional further usual ingredients, which are described below.
Preferred non-ionic surfactants are alkoxylated, advantageously
ethoxylated, particularly primary alcohols preferably containing 8
to 18 carbon atoms and, on average, 1 to 12 moles of ethylene oxide
(EO) per mole of alcohol, in which the alcohol group may be linear
or, preferably, methyl-branched in the 2-position or may contain
linear and methyl-branched groups in the form of the mixtures
typically present in oxoalcohol groups. In particular, however,
alcohol ethoxylates with linear alcohol groups of natural origin
with 12 to 18 carbon atoms, e.g. from coco-, palm-, tallow- or
oleyl alcohol, and an average of 2 to 8 EO per mole alcohol are
preferred. Exemplary preferred ethoxylated alcohols include
C.sub.12-14 alcohols with 3 EO or 4EO, C.sub.9-11 alcohols with 7
EO, C.sub.13-15 alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C.sub.12-18
alcohols with 3 EO, 5 EO or 7 EO and mixtures thereof, as well as
mixtures of C.sub.12-14 alcohol with 3 EO and C.sub.12-18 alcohol
with 5 EO. The cited degrees of ethoxylation constitute
statistically average values that can be a whole or a fractional
number for a specific product. Preferred alcohol ethoxylates have a
narrowed homolog distribution (narrow range ethoxylates, NRE). In
addition to these non-ionic surfactants, fatty alcohols with more
than 12 EO can also be used. Examples of these are tallow fatty
alcohol with 14 EO, 25 EO, 30 EO or 40 EO.
Another class of preferred non-ionic surfactants which may be used,
either as the sole non-ionic surfactant or in combination with
other non-ionic surfactants are alkoxylated, preferably ethoxylated
or ethoxylated and propoxylated fatty acid alkyl esters preferably
containing 1 to 4 carbon atoms in the alkyl chain, in particular
fatty acid methyl esters.
A further class of non-ionic surfactants, which can be
advantageously used, are the alkyl polyglycosides (APG). Suitable
alkyl polyglycosides satisfy the general Formula RO(G)z where R is
a linear or branched, particularly 2-methyl-branched, saturated or
unsaturated aliphatic group containing 8 to 22, preferably 12 to 18
carbon atoms and G is a symbol that stands for a glycose unit
containing 5 or 6 carbon atoms, preferably for glucose. Here, the
degree of glycosidation z is between 1.0 and 4.0, preferably
between 1.0 and 2.0 and particularly between 1.1 and 1.4. Linear
alkyl polyglucosides are preferably employed, that is alkyl
polyglycosides, in which the polyglycosyl group is a glucose group
and the alkyl group is an n-alkyl group.
Non-ionic surfactants of the amine oxide type, for example
N-cocoalkyl-N,N-dimethylamine oxide and N-tallow
alkyl-N,N-dihydroxyethylamine oxide, and the fatty acid
alkanolamides may also be suitable. The quantity of these non-ionic
surfactants is preferably no more than the quantity in which the
ethoxylated fatty alcohols are used and, particularly no more than
half that quantity.
Other suitable surfactants are polyhydroxyfatty acid amides
corresponding to the Formula (II),
##STR00001##
in which RCO stands for an aliphatic acyl group with 6 to 22 carbon
atoms, R.sup.1 for hydrogen, an alkyl or hydroxyalkyl group with 1
to 4 carbon atoms and [Z] for a linear or branched polyhydroxyalkyl
group with 3 to 10 carbon atoms and 3 to 10 hydroxyl groups. The
polyhydroxyfatty acid amides are known substances, which may
normally be obtained by reductive amination of a reducing sugar
with ammonia, an alkylamine or an alkanolamine and subsequent
acylation with a fatty acid, a fatty acid alkyl ester or a fatty
acid chloride.
The group of polyhydroxyfatty acid amides also includes compounds
corresponding to the Formula (III),
##STR00002##
in which R is a linear or branched alkyl or alkenyl group
containing 7 to 12 carbon atoms, R.sup.1 is a linear, branched or
cyclic alkyl group or an aryl group containing 2 to 8 carbon atoms
and R.sup.2 is a linear, branched or cyclic alkyl group or an aryl
group or an oxyalkyl group containing 1 to 8 carbon atoms,
C.sub.1-4 alkyl or phenyl groups being preferred, and [Z] is a
linear polyhydroxyalkyl group, of which the alkyl chain is
substituted by at least two hydroxyl groups, or alkoxylated,
preferably ethoxylated or propoxylated derivatives of that
group.
[Z] is preferably obtained by reductive amination of a sugar, for
example glucose, fructose, maltose, lactose, galactose, mannose or
xylose. The N-alkoxy- or N-aryloxy-substituted compounds may then
be converted into the required polyhydroxyfatty acid amides by
reaction with fatty acid methyl esters in the presence of an
alkoxide as catalyst.
Exemplary suitable anionic surfactants are those of the sulfonate
and sulfate type. Suitable surfactants of the sulfonate type are,
advantageously C.sub.9-13 alkylbenzene sulfonates, olefin
sulfonates, i.e. mixtures of alkene- and hydroxyalkane sulfonates,
and disulfonates, as are obtained, for example, from C.sub.12-18
monoolefins having a terminal or internal double bond, by
sulfonation with gaseous sulfur trioxide and subsequent alkaline or
acidic hydrolysis of the sulfonation products. Those alkane
sulfonates, obtained from C.sub.12-18 alkanes by sulfochlorination
or sulfoxidation, for example, with subsequent hydrolysis or
neutralization, are also suitable. The esters of .alpha.-sulfofatty
acids (ester sulfonates), e.g. the .alpha.-sulfonated methyl esters
of hydrogenated coco-, palm nut- or tallow acids are likewise
suitable.
Further suitable anionic surfactants are sulfated fatty acid esters
of glycerine. They include the mono-, di- and triesters and also
mixtures of them, such as those obtained by the esterification of a
monoglycerine with 1 to 3 moles fatty acid or the
transesterification of triglycerides with 0.3 to 2 moles glycerine.
Preferred sulfated fatty acid esters of glycerol in this case are
the sulfated products of saturated fatty acids with 6 to 22 carbon
atoms, for example caproic acid, caprylic acid, capric acid,
myristic acid, lauric acid, palmitic acid, stearic acid or behenic
acid.
Preferred alk(en)yl sulfates are the alkali metal and especially
sodium salts of the sulfuric acid half-esters derived from the
C.sub.12-C.sub.18 fatty alcohols, for example from coconut butter
alcohol, tallow alcohol, lauryl, myristyl, cetyl or stearyl alcohol
or from C.sub.10-C.sub.20 oxo alcohols and those half-esters of
secondary alcohols of these chain lengths. Additionally preferred
are alk(en)yl sulfates of the said chain lengths, which contain a
synthetic, straight-chained alkyl group produced on a petrochemical
basis and which show similar degradation behavior to the suitable
compounds based on fat chemical raw materials. The
C.sub.12-C.sub.16 alkyl sulfates and C.sub.12-C.sub.15 alkyl
sulfates and C.sub.14-C.sub.15 alkyl sulfates are preferred on the
grounds of laundry performance. 2,3-Alkyl sulfates are also
suitable anionic surfactants.
Sulfuric acid mono-esters derived from straight-chain or branched
C.sub.7-21 alcohols ethoxylated with 1 to 6 moles ethylene oxide
are also suitable, for example 2-methyl-branched C.sub.9-11
alcohols with an average of 3.5 mole ethylene oxide (EO) or
C.sub.12-18 fatty alcohols with 1 to 4 EO. Due to their high
foaming performance, they are only used in fairly small quantities
in cleaning compositions, for example in amounts of up to 5% by
weight, usually from 1 to 5% by weight.
Other suitable anionic surfactants are also the salts of
alkylsulfosuccinic acid, which are also referred to as
sulfosuccinates or esters of sulfosuccinic acid and the monoesters
and/or di-esters of sulfosuccinic acid with alcohols, preferably
fatty alcohols and especially ethoxylated fatty alcohols. Preferred
sulfosuccinates comprise C.sub.8-18 fatty alcohol groups or
mixtures of them. Especially preferred sulfosuccinates comprise a
fatty alcohol group derived from ethoxylated fatty alcohols and may
be considered as non-ionic surfactants (see description above).
Once again the especially preferred sulfosuccinates are those,
whose fatty alcohol groups are derived from ethoxylated fatty
alcohols with narrow range distribution. It is also possible to use
alk(en)ylsuccinic acids with preferably 8 to 18 carbon atoms in the
alk(en)yl chain, or salts thereof.
Soaps in particular can be considered as further anionic
surfactants. Saturated fatty acid soaps are suitable, such as the
salts of lauric acid, myristic acid, palmitic acid, stearic acid,
hydrogenated erucic acid and behenic acid, and especially soap
mixtures derived from natural fatty acids such as coconut oil fatty
acid, palm kernel oil fatty acid or tallow fatty acid.
Anionic surfactants, including soaps may be in the form of their
sodium, potassium or ammonium salts or as soluble salts of organic
bases, such as mono-, di- or triethanolamine. Preferably, the
anionic surfactants are in the form of their sodium or potassium
salts, especially in the form of the sodium salts.
The surfactants can be comprised in the inventive cleaning
compositions or detergents in an amount of preferably 5 to 50 wt.
%, particularly 8 to 30 wt. %, based on the finished
composition.
The inventive detergents or cleaning agents can comprise bleaching
agent. Among the compounds, which serve as bleaches and liberate
H.sub.2O.sub.2 in water, sodium percarbonate, sodium perborate
tetrahydrate and sodium perborate monohydrate are of particular
importance. Examples of further bleaching agents, which may be
used, are peroxypyrophosphates, citrate perhydrates and
H.sub.2O.sub.2-liberating peracidic salts or peracids, such as
persulfates or persulfuric acid. The urea peroxyhydrate
percarbamide that can be described by the formula
H.sub.2N--CO--NH.sub.2.H.sub.2O.sub.2 is also suitable.
Particularly when agents are used to clean hard surfaces, for
example in automatic dishwashers, they can, if desired, also
comprise bleaching agents from the group of the organic bleaching
agents, although in principal they can also be used for washing
textiles. Typical organic bleaching agents are the diacyl
peroxides, such as e.g. dibenzoyl peroxide. Further typical organic
bleaching agents are the peroxy acids, wherein the alkylperoxy
acids and the arylperoxy acids may be named as examples. Preferred
representatives that can be added are peroxybenzoic acid and
ring-substituted derivatives thereof, such as alkyl peroxybenzoic
acids, but also peroxy-.alpha.-naphthoic acid and magnesium
monoperphthalate, the aliphatic or substituted aliphatic peroxy
acids, such as peroxylauric acid, peroxystearic acid,
.epsilon.-phthalimidoperoxycaproic acid [phthaloiminoperoxyhexanoic
acid, PAP)], o-carboxybenzamidoperoxycaproic acid, N-nonenylamido
peradipic acid and N-nonenylamido persuccinates and aliphatic and
araliphatic peroxydicarboxylic acids, such as
1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid,
diperoxysebacic acid, diperoxybrassylic acid, diperoxyphthalic
acids, 2-decyldiperoxybutane-1,4-dioic acid,
N,N-terephthaloyl-di(6-aminopercaproic acid).
The bleaching agent content of the detergent or cleaning agent is
preferably 1 to 40 wt. % and particularly 10 to 20 wt. %, perborate
monohydrate or percarbonate being advantageously used.
The preparations can also comprise bleach activators in order to
achieve an improved bleaching action for washing temperatures of
60.degree. C. and below and particularly during the pre-treatment
wash. Bleach activators, which can be used are compounds which,
under perhydrolysis conditions, yield aliphatic peroxycarboxylic
acids having preferably 1 to 10 carbon atoms, in particular 2 to 4
carbon atoms, and/or optionally substituted perbenzoic acid.
Substances, which carry O-acyl and/or N-acyl groups of said number
of carbon atoms and/or optionally substituted benzoyl groups, are
suitable. Preference is given to polyacylated alkylenediamines, in
particular tetraacetyl ethylene diamine (TAED), acylated triazine
derivatives, in particular
1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated
glycolurils, in particular tetraacetyl glycoluril (TAGU),
N-acylimides, in particular N-nonanoyl succinimide (NOSI), acylated
phenol sulfonates, in particular n-nonanoyl- or
isononanoyloxybenzene sulfonate (n- or iso-NOBS), carboxylic acid
anhydrides, in particular phthalic anhydride, acylated polyhydric
alcohols, in particular triacetin, ethylene glycol diacetate and
2,5-diacetoxy-2,5-dihydrofuran and the enol esters known from the
German Patent applications DE 196 16 693 and DE 196 16 767 and
acetylated sorbitol and mannitol or their mixtures (SORMAN)
described in the European Patent application EP-A-767 0 525,
acylated sugar derivatives, in particular pentaacetylglucose (PAG),
pentaacetylfructose, tetraacetylxylose and octaacetyllactose as
well as acetylated, optionally N-alkylated glucamine and
gluconolactone, triazole or triazole derivatives and/or particulate
caprolactams and/or caprolactam derivatives, preferably N-acylated
lactams, for example N-benzoylcaprolactam and N-acetylcaprolactam.
Hydrophilically substituted acyl acetals and acyl lactams are also
preferably used. Combinations of conventional bleach activators may
also be used. Nitrile derivatives such as cyanopyridines,
nitrilequats, for example N-alkyl ammonium acetonitrile, and/or
cyanamide derivatives can also be used. Preferred bleach activators
are sodium 4-(octanoyloxy)benzene sulfonate, n-nonanoyl- or
isononanoyloxybenzene sulfonate (n- or iso-NOBS),
undecenoyloxybenzene sulfonate (UDOBS), sodium dodecanoyloxybenzene
sulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC 10) and/or
dodecanoyloxybenzene sulfonate (OBS 12), and N-methylmorpholinum
acetonitrile (MMA). These types of bleach activators are comprised
in the usual quantity range of 0.01 to 20 wt. %, preferably in
amounts of 0.1 wt. % to 15 wt. %, particularly 1 wt. % to 10 wt. %,
based on the total composition.
In addition to, or instead of the conventional bleach activators
mentioned above, so-called bleach catalysts may also be
incorporated. These substances are bleach-boosting transition metal
salts or transition metal complexes such as, for example,
manganese-, iron-, cobalt-, ruthenium- or molybdenum-salen or
-carbonyl complexes. Manganese, iron, cobalt, ruthenium,
molybdenum, titanium, vanadium and copper complexes with
nitrogen-containing tripod ligands, as well as cobalt-, iron-,
copper- and ruthenium-ammine complexes may also be employed as the
bleach catalysts, wherein those compounds that are described in DE
197 09 284 A1 are preferably employed.
Generally, inventive detergents or cleaning agents comprise one or
more builders, in particular zeolites, silicates, carbonates,
organic cobuilders and--where there are no ecological grounds
against their use--also phosphates. The last are particularly
preferred builders employed in cleaning compositions for automatic
dishwashers.
Suitable silicate builders are the crystalline, layered sodium
silicates corresponding to the general formula
NaMSi.sub.xO.sub.2x+1yH.sub.2O, wherein M is sodium or hydrogen, x
is a number from 1.6 to 4, preferably 1.9 to 4.0 and y is a number
from 0 to 20, preferred values for x being 2, 3 or 4. These types
of crystalline layered silicates are described, for example, in the
patent literature. Preferred crystalline layered silicates of the
given formula are those in which M stands for sodium and x assumes
the values 2 or 3. Both .beta.- and also .delta.-sodium disilicates
Na.sub.2Si.sub.2O.sub.5yH.sub.2O are particularly preferred. These
types of compounds are commercially available, for example, under
the designation SKS.RTM. (Clariant). SKS-6.RTM. is mainly a
.delta.-sodium disilicate with the formula
Na.sub.2Si.sub.2O.sub.5yH.sub.2O and SKS-7.RTM. is mainly the
.beta.-sodium disilicate. On reaction with acids (e.g. citric acid
or carbonic acid), .delta.-sodium silicate affords Kanemit
NaHSi.sub.2O.sub.5yH.sub.2O, commercially available under the trade
names SKS-9.RTM. and SKS-10.RTM. (Clariant). It can also be
advantageous to chemically modify these layered silicates. The
alkalinity, for example, of the layered silicates can be suitably
modified. In comparison with the 5-sodium disilicate, layered
silicates, doped with phosphate or carbonate, exhibit a different
crystal morphology, dissolve more rapidly and show an increased
calcium binding ability. Thus, layered silicates of the general
formula x Na.sub.2O y SiO.sub.2 z P.sub.2O.sub.5 in which the ratio
x to y corresponds to a number 0.35 to 0.6, the ratio x to z a
number from 1.75 to 1200 and the ratio y to z a number from 4 to
2800, are described in the patent application DE 196 01 063. The
solubility of the layered silicates can also be increased by
employing particularly finely dispersed layered silicates.
Compounds of the crystalline layered silicates with other
ingredients can also be used. Compounds with cellulose derivatives,
which possess advantages in the disintegration action, and which
are particularly used in detergent tablets, as well as compounds
with polycarboxylates, for example citric acid or polymeric
polycarboxylates, for example copolymers of acrylic acid can be
particularly cited in this context.
Other useful builders are amorphous sodium silicates with a modulus
(Na.sub.2O:SiO.sub.2 ratio) of 1:2 to 1:3.3, preferably 1:2 to
1:2.8 and more preferably 1:2 to 1:2.6, which dissolve with a delay
and exhibit multiple wash cycle properties. The delay in
dissolution compared with conventional amorphous sodium silicates
can have been obtained in various ways, for example by surface
treatment, compounding, compressing/compacting or by over-drying.
In the context of this invention, the term "amorphous" also means
"X-ray amorphous". In other words, the silicates do not produce any
of the sharp X-ray reflections typical of crystalline substances,
but at best one or more maxima of the scattered X-radiation, which
have a width of several degrees of the diffraction angle. However,
particularly good builder properties may even be achieved where the
silicate particles produce indistinct or even sharp diffraction
maxima in electron diffraction experiments. This is interpreted to
mean that the products have microcrystalline regions between 10 and
a few hundred nm in size, values of up to at most 50 nm and
especially up to at most 20 nm being preferred. Compacted/densified
amorphous silicates, compounded amorphous silicates and over dried
X-ray-amorphous silicates are particularly preferred.
An optionally suitable fine crystalline, synthetic zeolite
containing bound water, is preferably zeolite A and/or P. Zeolite
MAP.RTM. (commercial product of the Crosfield company), is
particularly preferred as the zeolite P. However, zeolite X and
mixtures of A, X and/or P are also suitable. Commercially available
and preferably used in the context of the present invention is, for
example, also a co-crystallizate of zeolite X and zeolite A (ca. 80
wt. % zeolite X), which is marketed by CONDEA Augusta S.p.A. under
the trade name VEGOBOND AX.RTM. and which can be described by the
Formula
nNa.sub.2O(1-n)K.sub.2OAl.sub.2O.sub.3(2-2.5)SiO.sub.2(3.5-5.5)H.sub.2O
Suitable zeolites have an average particle size of less than 10
.mu.m (test method: volumetric distribution Coulter counter) and
preferably comprise 18 to 22 wt. %, particularly 20 to 22 wt. % of
bound water.
Naturally, the generally known phosphates can also be added as
builders, in so far that their use should not be avoided on
ecological grounds. In the detergent and cleaning agent industry,
among the many commercially available phosphates, the alkali metal
phosphates are the most important and pentasodium or pentapotassium
triphosphates (sodium or potassium tripolyphosphate) are
particularly preferred.
"Alkali metal phosphates" is the collective term for the alkali
metal (more particularly sodium and potassium) salts of the various
phosphoric acids, in which metaphosphoric acids (HPO3)n and
orthophosphoric acid (H3PO4) and representatives of higher
molecular weight can be differentiated. The phosphates combine
several inherent advantages: they act as alkalinity sources,
prevent lime deposits on machine parts and lime incrustations in
fabrics and, in addition, contribute towards the cleaning
effect.
Sodium dihydrogen phosphate NaH.sub.2PO.sub.4 exists as the
dihydrate (density 1.91 gcm-3, melting point 60.degree. C.) and as
the monohydrate (density 2.04 gcm-3). Both salts are white, readily
water-soluble powders that on heating, lose the water of
crystallization and at 200.degree. C. are converted into the weakly
acidic diphosphate (disodium hydrogen diphosphate,
Na.sub.2H.sub.2P.sub.2O.sub.7) and, at higher temperatures into
sodium trimetaphosphate (Na.sub.3P.sub.3O.sub.9) and Maddrell's
salt (see below). NaH.sub.2PO.sub.4 shows an acidic reaction. It is
formed by adjusting phosphoric acid with sodium hydroxide to a pH
value of 4.5 and spraying the resulting "mash". Potassium
dihydrogen phosphate (primary or monobasic potassium phosphate,
potassium biphosphate, KDP), KH.sub.2PO.sub.4, is a white salt with
a density of 2.33 gcm-3, has a melting point of 253.degree. C.
[decomposition with formation of potassium polyphosphate
(KPO.sub.3).sub.x] and is readily soluble in water.
Disodium hydrogen phosphate (secondary sodium phosphate),
Na.sub.2HPO.sub.4, is a colorless, very readily water-soluble
crystalline salt. It exists in anhydrous form and with 2 mol
(density 2.066 gcm-3, water loss at 95.degree. C.), 7 mol (density
1.68 gcm-3, melting point 48.degree. C. with loss of 5H.sub.2O) and
12 mol of water (density 1.52 gcm-3, melting point 35.degree. C.
with loss of 5H.sub.2O), becomes anhydrous at 100.degree. C. and,
on fairly intensive heating, is converted into the diphosphate
Na.sub.4P.sub.2O.sub.7. Disodium hydrogen phosphate is prepared by
neutralization of phosphoric acid with soda solution using
phenolphthalein as the indicator. Dipotassium hydrogen phosphate
(secondary or dibasic potassium phosphate), K.sub.2HPO.sub.4, is an
amorphous white salt, which is readily soluble in water.
Trisodium phosphate, tertiary sodium phosphate, Na.sub.3PO.sub.4,
consists of colorless crystals that as the dodecahydrate have a
density of 1.62 gcm-3 and a melting point of 73-76.degree. C.
(decomposition), as the decahydrate (corresponding to 19-20%
P.sub.2O.sub.5) a melting point of 100.degree. C. and in anhydrous
form (corresponding to 39-40% P.sub.2O.sub.5) a density of 2.536
gcm-3. Trisodium phosphate is readily soluble in water with an
alkaline reaction and is manufactured by evaporating a solution of
exactly 1 mole disodium phosphate and 1 mole NaOH. Tripotassium
phosphate (tertiary or tribasic potassium phosphate),
K.sub.3PO.sub.4, is a white deliquescent granular powder with a
density of 2.56 gcm-3, has a melting point of 1340.degree. C. and
is readily soluble in water through an alkaline reaction. It is
produced by e.g. heating Thomas slag with carbon and potassium
sulfate. Despite their higher price, the more readily soluble and
therefore highly effective potassium phosphates are often preferred
to corresponding sodium compounds in the detergent industry.
Tetrasodium diphosphate (sodium pyrophosphate),
Na.sub.4P.sub.2O.sub.7, exists in anhydrous form (density 2.534
gcm-3, melting point 988.degree. C., a figure of 880.degree. C. has
also been mentioned) and as the decahydrate (density 1.815-1.836
gcm-3, melting point 94.degree. C. with loss of water). Both
substances are colorless crystals that dissolve in water with an
alkaline reaction. Na.sub.4P.sub.2O.sub.7 is formed when disodium
phosphate is heated to more than 200.degree. C. or by reacting
phosphoric acid with soda in a stoichiometric ratio and spray
drying the solution. The decahydrate complexes heavy metal salts
and hardness salts and, hence, reduces the hardness of water.
Potassium diphosphate (potassium pyrophosphate),
K.sub.4P.sub.2O.sub.7, exists in the form of the trihydrate and is
a colorless hygroscopic powder with a density of 2.33 gcm-3, which
is soluble in water, the pH of a 1% solution at 25.degree. C. being
10.4.
Relatively high molecular weight sodium and potassium phosphates
are formed by condensation of NaH.sub.2PO.sub.4 or
KH.sub.2PO.sub.4. They may be divided into cyclic types, namely the
sodium and potassium metaphosphates, and chain types, the sodium
and potassium polyphosphates. The chain types in particular are
known by various different names: fused or calcined phosphates,
Graham's salt, Kurrol's salt and Maddrell's salt. All higher sodium
and potassium phosphates are known collectively as condensed
phosphates.
The industrially important pentasodium triphosphate,
Na.sub.5P.sub.3O.sub.10 (sodium tripolyphosphate), is anhydrous or
crystallizes with 6H.sub.2O to a non-hygroscopic, white,
water-soluble salt which has the general formula
NaO--[P(O)(ONa)--O]n-Na where n.dbd.3. Around 17 g of the salt free
from water of crystallization dissolve in 100 g of water at room
temperature, around 20 g at 60.degree. C. and around 32 g at
100.degree. C. After heating the solution for 2 hours to
100.degree. C., around 8% orthophosphate and 15% diphosphate are
formed by hydrolysis. In the preparation of pentasodium
triphosphate, phosphoric acid is reacted with soda solution or
sodium hydroxide in a stoichiometric ratio and the solution is
spray-dried. Similarly to Graham's salt and sodium diphosphate,
pentasodium triphosphate solubilizes many insoluble metal compounds
(including lime soaps, etc.). K.sub.5P.sub.3O.sub.10 (potassium
tripolyphosphate), is marketed for example in the form of a 50% by
weight solution (>23% P.sub.2O.sub.5, 25% K.sub.2O). The
potassium polyphosphates are widely used in the detergent industry.
Sodium potassium tripolyphosphates also exist and are also usable
in the scope of the present invention. They are formed for example
when sodium trimetaphosphate is hydrolyzed with KOH:
(NaPO.sub.3).sub.3+2KOH->Na.sub.3K.sub.2P.sub.3O.sup.10+H.sub.2O
According to the invention, they may be used in exactly the same
way as sodium tripolyphosphate, potassium tripolyphosphate or
mixtures thereof. Mixtures of sodium tripolyphosphate and sodium
potassium tripolyphosphate or mixtures of potassium
tripolyphosphate and sodium potassium tripolyphosphate or mixtures
of sodium tripolyphosphate and potassium tripolyphosphate and
sodium potassium tripolyphosphate may also be used in accordance
with the invention.
Organic co builders, which may be used in the detergents and
cleaning agents according to the invention, include, in particular,
polycarboxylates or polycarboxylic acids, polymeric
polycarboxylates, polyaspartic acid, polyacetals, optionally
oxidized dextrins, other organic co builders (see below) and
phosphonates. These classes of substances are described below.
Useful organic builders are, for example, the polycarboxylic acids
usable in the form of their sodium salts, polycarboxylic acids in
this context being understood to be carboxylic acids that carry
more than one acid function. These include, for example, citric
acid, adipic acid, succinic acid, glutaric acid, malic acid,
tartaric acid, maleic acid, fumaric acid, sugar acids,
aminocarboxylic acids, nitrilotriacetic acid (NTA), providing its
use is not ecologically unsafe, and mixtures thereof. Preferred
salts are the salts of polycarboxylic acids such as citric acid,
adipic acid, succinic acid, glutaric acid, tartaric acid, sugar
acids and mixtures thereof.
Acids per se can also be used. Besides their building effect, the
acids also typically have the property of an acidifying component
and, hence also serve to establish a relatively low and mild pH in
washing or cleaning agents, when the pH, which results from the
mixture of other components, is not wanted. Acids that are
system-compatible and environmentally compatible such as citric
acid, acetic acid, tartaric acid, malic acid, glycolic acid,
succinic acid, glutaric acid, adipic acid, gluconic acid and
mixtures thereof are particularly mentioned in this regard.
However, mineral acids, particularly sulfuric acid or bases,
particularly ammonium or alkali metal hydroxides can also serve as
pH regulators. These types of regulators are preferably comprised
in the inventive agents in amounts of not more than 20 wt. %,
particularly from 1.2 wt. % to 17 wt. %.
Other suitable builders are polymeric polycarboxylates, i.e. for
example the alkali metal salts of polyacrylic or polymethacrylic
acid, for example those with a relative molecular weight of 500 to
70 000 g/mol.
The molecular weights mentioned in this specification for polymeric
polycarboxylates are weight-average molecular weights Mw of the
particular acid form which, fundamentally, were determined by gel
permeation chromatography (GPC), equipped with a UV detector. The
measurement was carried out against an external polyacrylic acid
standard, which provides realistic molecular weight values by
virtue of its structural similarity to the polymers investigated.
These values differ significantly from the molecular weights
measured against polystyrene sulfonic acids as the standard. The
molecular weights measured against polystyrene sulfonic acids are
generally significantly higher than the molecular weights mentioned
in this specification.
Particularly suitable polymers are polyacrylates, which preferably
have a molecular weight of 2000 to 20 000 g/mol. By virtue of their
superior solubility, preferred representatives of this group are
the short-chain polyacrylates, which have molecular weights of 2000
to 10 000 g/mol and, more particularly, 3000 to 5000 g/mol.
Further suitable copolymeric polycarboxylates are particularly
those of acrylic acid with methacrylic acid and of acrylic acid or
methacrylic acid with maleic acid. Copolymers of acrylic acid with
maleic acid, which comprise 50 to 90 wt. % acrylic acid and 50 to
10 wt. % maleic acid, have proven to be particularly suitable.
Their relative molecular weight, based on free acids, generally
ranges from 2 000 to 70 000 g/mol, preferably 20 000 to 50 000
g/mol and especially 30 000 to 40 000 g/mol. The (co)polymeric
polycarboxylates can be used either as powders or as aqueous
solutions. The (co)polymeric polycarboxylate content of the
compositions is preferably from 0.5 to 20% by weight, in particular
from 1 to 10% by weight.
In order to improve the water solubility, the polymers can also
comprise allylsulfonic acids, such as for example, allyloxybenzene
sulfonic acid and methallyl sulfonic acid as monomers.
Other particularly preferred polymers are biodegradable polymers of
more than two different monomer units, for example those which
contain salts of acrylic acid and maleic acid and vinyl alcohol or
vinyl alcohol derivatives as monomers or those which contain salts
of acrylic acid and 2-alkylallyl sulfonic acid and sugar
derivatives as monomers.
Other preferred copolymers are those, which preferably contain
acrolein and acrylic acid/acrylic acid salts or acrolein and vinyl
acetate as monomers.
Similarly, other preferred builders are polymeric amino
dicarboxylic acids, salts or precursors thereof. Polyaspartic acids
or their salts and derivatives are particularly preferred.
Further preferred builders are polyacetals that can be obtained by
treating dialdehydes with polyol carboxylic acids that possess 5 to
7 carbon atoms and at least 3 hydroxyl groups. Preferred
polyacetals are obtained from dialdehydes like glyoxal,
glutaraldehyde, terephthalaldehyde as well as their mixtures and
from polycarboxylic acids like gluconic acid and/or glucoheptonic
acid.
Further suitable organic builders are dextrins, for example
oligomers or polymers of carbohydrates that can be obtained by the
partial hydrolysis of starches. The hydrolysis can be carried out
using typical processes, for example acidic or enzymatic catalyzed
processes. The hydrolysis products preferably have average
molecular weights in the range 400 to 500 000 g/mol. A
polysaccharide with a dextrose equivalent (DE) of 0.5 to 40 and,
more particularly, 2 to 30 is preferred, the DE being an accepted
measure of the reducing effect of a polysaccharide by comparison
with dextrose, which has a DE of 100. Both maltodextrins with a DE
between 3 and 20 and dry glucose syrups with a DE between 20 and 37
and also so-called yellow dextrins and white dextrins with
relatively high molecular weights of 2000 to 30 000 g/mol may be
used.
The oxidized derivatives of such dextrins concern their reaction
products with oxidizing agents that are capable of oxidizing at
least one alcohol function of the saccharide ring to the carboxylic
acid function. Particularly preferred organic builders for
inventive agents are oxidized starches or their derivatives.
Oxydisuccinates and other derivatives of disuccinates, preferably
ethylenediamine disuccinate are also further suitable cobuilders.
Ethylenediamine-N,N'-disuccinate (EDDS) is preferably used here in
the form of its sodium or magnesium salts. In this context,
glycerine disuccinates and glycerine trisuccinates are also
preferred. Suitable addition quantities in zeolite-containing
and/or silicate-containing formulations range between 3 and 15% by
weight.
Other useful organic co-builders are, for example, acetylated
hydroxycarboxylic acids and salts thereof which may optionally be
present in lactone form and which contain at least 4 carbon atoms,
at least one hydroxy group and at most two acid groups.
The phosphonates represent a further class of substances with
cobuilder properties. In particular, they are hydroxyalkane
phosphonates or aminoalkane phosphonates. Among the hydroxyalkane
phosphonates, 1-hydroxyethane-1,1-diphosphonate (HEDP) is of
particular importance as the cobuilder. It is normally added as the
sodium salt, the disodium salt reacting neutral and the tetrasodium
salt reacting alkaline (pH 9). Ethylenediamine tetramethylene
phosphonate (EDTMP), diethylenetriamine pentamethylene phosphonate
(DTPMP) and their higher homologs are preferably chosen as the
aminoalkane phosphonates. They are preferably added in the form of
the neutral-reacting sodium salts, e.g. as the hexasodium salt of
EDTMP or as the hepta and octasodium salt of DTPMP. Of the class of
phosphonates, HEDP is preferably used as the builder. The
aminoalkane phosphonates additionally possess a pronounced ability
to complex heavy metals. Accordingly, it can be preferred,
particularly where the agents also contain bleach, to use
aminoalkane phosphonates, particularly DTPMP, or mixtures of the
mentioned phosphonates.
In addition, any compounds capable of forming complexes with
alkaline earth metal ions may be used as co-builders.
Builders can be comprised in the inventive detergents or cleaning
agents optionally in quantities of up to 90% by weight. They are
preferably comprised in quantities of up to 75% by weight.
Inventive detergents possess builder contents of particularly 5 wt.
% to 50 wt. %. In inventive compositions for cleaning hard
surfaces, in particular for automatic dishwashing of tableware, the
content of builders is particularly 5 wt. % to 88 wt. %, wherein in
this type of composition, no water-insoluble builders are employed.
In a preferred embodiment, the inventive agent, particularly for
automatic dishwashing of tableware, comprises 20 wt. % to 40 wt. %
of water-soluble organic builders, particularly alkali citrate, 5
wt. % to 15 wt. % alkali carbonate and 20 wt. % to 40 wt. % alkali
disilicate.
Solvents that can be added to the liquid to gel-like compositions
of detergents and cleaning agents originate, for example, from the
group of mono- or polyhydric alcohols, alkanolamines or glycol
ethers, in so far that they are miscible with water in the defined
concentrations. Preferably, the solvents are selected from ethanol,
n- or i-propanol, butanols, ethylene glycol methyl ether, ethylene
glycol ethyl ether, ethylene glycol propyl ether, ethylene glycol
mono-n-butyl ether, diethylene glycol methyl ether, diethylene
glycol ethyl ether, propylene glycol methyl-, -ethyl- or -propyl
ether, dipropylene glycol methyl-, or -ethyl ether, methoxy-,
ethoxy- or butoxy triglycol, 1-butoxyethoxy-2-propanol,
3-methyl-3-methoxybutanol, propylene glycol t-butyl ether as well
as mixtures of these solvents.
Solvents can be employed in the inventive liquid to gel-like
detergents and cleaning compositions in amounts between 0.1 and 20
wt. %, preferably, however below 15 wt. % and particularly below 10
wt. %.
One or more thickeners or thickener systems can be added to the
inventive compositions to adjust the viscosity. These high
molecular weight substances, which are also called swelling agents,
soak up mostly liquids, thereby swelling up and subsequently
transform into viscous, real or colloidal solutions.
Suitable thickeners are inorganic or polymeric organic compounds.
The inorganic thickeners include, for example, polysilicic acids,
mineral clays like montmorillonite, zeolites, silicic acids and
bentonites. The organic thickeners come from the groups of natural
polymers, derivatives of natural polymers and synthetic polymers.
Exemplary, naturally occurring polymers that can be used as
thickeners are agar agar, carrageen, tragacanth, gum Arabic,
alginates, pectins, polyoses, guar meal, locust tree bean flour,
starches, dextrins, gelatines and casein. Modified natural products
that are used as thickeners are mainly derived from the group of
the modified starches and celluloses. Examples can be cited as
carboxymethyl cellulose and other cellulose ethers, hydroxyethyl-
and hydroxypropyl cellulose as well as flour ether. Totally
synthetic thickeners are polymers such as polyacrylics and
polymethacrylics, vinyl polymers, polycarboxylic acids, polyethers,
polyimines, polyamides and polyurethanes.
The thickeners can be comprised in amounts up to 5 wt. %,
preferably from 0.05 to 2 wt. %, and particularly preferably from
0.1 to 1.5 wt. %, based on the finished preparation.
The detergents or cleaning agents according to the invention can
optionally comprise further typical ingredients--sequestering
agents, electrolytes and further auxiliaries, such as optical
brighteners, redeposition inhibitors, silver corrosion inhibitors,
color transfer inhibitors, foam inhibitors, abrasives, dyes and/or
fragrances, as well as antimicrobial agents UV absorbers and/or
enzyme stabilizers.
The detergents for textiles may contain derivatives of
diaminostilbene disulfonic acid or alkali metal salts thereof as
optical brighteners. Suitable optical brighteners are, for example,
salts of
4,4'-bis-(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene-2,2'-di-
sulfonic acid or compounds of similar structure which contain a
diethanolamino group, a methylamino group and anilino group or a
2-methoxyethylamino group instead of the morpholino group. Optical
brighteners of the substituted diphenylstyryl type may also be
present, for example the alkali metal salts of
4,4'-bis(2-sulfostyryl)diphenyl, 4,4'-bis(4-chloro-3-sulfostyryl)di
phenyl or 4-(4-chlorostyryl)-4'-(2-sulfostyryl)diphenyl. Mixtures
of the mentioned optical brighteners may also be used.
Graying inhibitors have the task of ensuring that the dirt removed
from the textile fibers is held suspended in the wash liquid.
Water-soluble colloids of mostly organic nature are suitable for
this, for example starch, glue, gelatines, salts of ether
carboxylic acids or ether sulfonic acids of starches or celluloses,
or salts of acidic sulfuric acid esters of celluloses or starches.
Water-soluble, acid group-containing polyamides are also suitable
for this purpose. Moreover, aldehyde starches, for example, can be
used instead of the abovementioned starch derivatives. Preference,
however, is given to the use of cellulose ethers such as
carboxymethyl cellulose (Na salt), methyl cellulose, hydroxyalkyl
cellulose, and mixed ethers such as methyl hydroxyethyl cellulose,
methyl hydroxypropyl cellulose, methyl carboxymethyl cellulose and
mixtures thereof, which can be added, for example in amounts of 0.1
to 5 wt. %, based on the agent.
In order to realize a silver corrosion protection, silver
protectors for tableware can be added to the inventive cleaning
agents. Benzotriazoles, ferric chloride or CoSO4, for example are
known from the prior art. As is known from the European Patent EP 0
736 084 B1, for example, particularly suitable silver corrosion
inhibitors for general use with enzymes are salts and/or complexes
of manganese, titanium, zirconium, hafnium, vanadium, cobalt or
cerium, in which the cited metals exist in the valence states II,
III, IV, V or VI. Examples of these types of compounds are
MnSO.sub.4, V.sub.2O.sub.5, V.sub.2O.sub.4, VO2, TiOSO.sub.4,
K.sub.2TiF.sub.6, K.sub.2ZrF.sub.6, Co(NO.sub.3).sub.2,
Co(NO.sub.3).sub.3 and mixtures thereof.
Soil repellents are mostly polymers that when used in a detergent,
lend the fibers soil repelling properties and/or support the soil
repellent capabilities of the conventional ingredients. A
comparable effect can also be observed when they are added in
cleaning compositions for hard surfaces.
Particularly effective and well-known soil release agents are
copolyesters with dicarboxylic acid, alkylene glycol and
polyalkylene glycol units. Examples of these are copolymers or
mixed polymers of polyethylene terephthalates and polyoxyethylene
glycol (DT 16 17 141 and DT 22 00 911). German Offenlegungsschrift
DT 22 53 063 cites acidic compositions, which inter alia comprise a
copolymer of a dibasic acid and an alkylene or cycloalkylene
polyglycol. Polymers of ethylene terephthalate and polyethylene
oxide-terephthalate and their use in detergents are described in
the prior art, likewise agents that comprise a copolyester of
ethylene glycol, polyethylene glycol, aromatic dicarboxylic acids
and sulfonated aromatic dicarboxylic acids in defined molar ratios.
Methyl or ethyl group end blocked polyesters with ethylene and or
propylene terephthalate and polyethylene oxide terephthalate units,
and detergents that comprise this type of soil release polymer are
known, as is polyester that in addition to oxyethylene groups and
terephthalic acid units also comprises substituted ethylene units
as well as glycerine units, and polyesters that in addition to
oxyethylene groups and terephthalic acid units comprise
2-propylene-, 1,2-butylene- and/or 3-methoxy-1,2-propylene groups
as well as glycerine units, and whose end groups are blocked with
C.sub.1 to C.sub.4 alkyl groups. At least partially C.sub.1-4
alkyl- or acyl end blocked polyesters containing polypropylene
terephthalate and polyoxyethylene terephthalate units, sulfoethyl
end blocked terephthalate-containing soil release polyester,
sulfonated unsaturated end group soil release polyesters containing
terephthalate, alkylene glycol and poly C.sub.2-C.sub.4 glycol
units and acidic aromatic soil release polyesters are known. In
addition, non-polymeric soil repellents for materials made of
cotton are known with a plurality of functional units: a first
unit, which can be cationic, for example, is able to be adsorbed
onto the cotton surface by electrostatic attraction, and a second
unit, which is designed to be hydrophobic, is responsible for the
retention of the active agent at the water/cotton interface.
Color transfer inhibitors that can be used in inventive detergents
for textiles particularly include polyvinyl pyrrolidones, polyvinyl
imidazoles, polymeric N-oxides, such as polyvinyl pyridine-N-oxide,
and copolymers of vinyl pyrrolidone with vinyl imidazole.
On using the agents in automatic cleaning processes, it can be
advantageous to add foam inhibitors. Suitable foam inhibitors
include for example, soaps of natural or synthetic origin, which
have a high content of C.sub.18-C.sub.24 fatty acids. Suitable
non-surface-active types of foam inhibitors are, for example,
organopolysiloxanes and mixtures thereof with microfine, optionally
silanized silica and also paraffins, waxes, microcrystalline waxes
and mixtures thereof with silanized silica or bis-stearyl ethylene
diamide. Mixtures of various foam inhibitors, for example mixtures
of silicones, paraffins or waxes, are also used with advantage.
Preferably, the foam inhibitors, especially silicone-containing
and/or paraffin-containing foam inhibitors, are loaded onto a
granular, water-soluble or dispersible carrier material. Especially
in this case, mixtures of paraffins and bis stearylethylene
diamides are preferred.
In processes for cleaning textiles or hard surfaces, the esterases
are employed in a quantity of 0.04 .mu.g to 96 g, preferably from
0.05 .mu.g to 72 g, particularly preferably from 1 .mu.g to 48 g
and quite particularly preferably from 2 .mu.g to 24 g per
application.
An inventive cleaning composition for hard surfaces can moreover
comprise abrasive ingredients, especially from the group comprising
quartz meal, wood flour, plastic powder, chalk and microspheres as
well as their mixtures. Abrasives are preferably comprised in the
inventive cleaning compositions in amounts of not more than 20 wt.
%, particularly from 5 wt. % to 15 wt. %.
Colorants and fragrances may be added to the detergents and
cleaning agents in order to improve the esthetic impression created
by the products and to provide the consumer not only with the
required performance but also with a visually and sensorially
"typical and unmistakable" product. Suitable perfume oils or
fragrances include individual perfume compounds, for example
synthetic products of the ester, ether, aldehyde, ketone, alcohol
and hydrocarbon type. Perfume compounds of the ester type are, for
example, benzyl acetate, phenoxyethyl isobutyrate,
p-tert.-butylcyclohexyl acetate, linalyl acetate, dimethylbenzyl
carbinyl acetate, phenylethyl acetate, linalyl benzoate, benzyl
formate, ethylmethylphenyl glycinate, allylcyclohexyl propionate,
styrallyl propionate and benzyl salicylate. The ethers include, for
example, benzyl ethyl ether; the aldehydes include, for example,
the linear alkanals containing 8 to 18 carbon atoms, citral,
citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde,
hydroxycitronellal, lilial and bourgeonal; the ketones include, for
example, the ionones, .alpha.-isomethyl ionone and methyl cedryl
ketone; the alcohols include anethol, citronellol, eugenol,
geraniol, linalool, phenylethyl alcohol and terpineol and the
hydrocarbons include, above all, the terpenes, such as limonene and
pinene. However, mixtures of various odoriferous substances, which
together produce an attractive fragrant note, are preferably used.
Perfume oils such as these may also contain natural odoriferous
mixtures obtainable from vegetal sources, for example pine, citrus,
jasmine, patchouli, rose or ylang-ylang oil. Also suitable are
muscatel oil, oil of sage, chamomile oil, clove oil, melissa oil,
mint oil, cinnamon leaf oil, lime blossom oil, juniper berry oil,
vetivert oil, olibanum oil, galbanum oil and laudanum oil and
orange blossom oil, neroli oil, orange peel oil and sandalwood oil.
Normally the content of dyes lies below 0.01 wt. %, while
fragrances can make up to 2 wt. % of the total formulation of the
detergents and cleaning composition.
The fragrances may be directly incorporated in the detergent or
cleaning agent, although it can also be of advantage to apply the
fragrances on carriers, which reinforce the adsorption of the
perfume on the washing and thereby ensuring a long-lasting
fragrance on the textiles by decreasing the release of the
fragrance, especially for treated textiles. Suitable carrier
materials are, for example, cyclodextrins, the cyclodextrin/perfume
complexes optionally being coated with other auxiliaries. A further
preferred carrier for fragrances is the described zeolite X, which
instead of or in mixtures with surfactants can also take up
fragrances. Accordingly, preferred detergents and cleaning agents
comprise the described zeolite X and fragrances that are preferably
at least partially absorbed on the zeolite.
Preferred colorants, which are not difficult for the expert to
choose, have high storage stability, are not affected by the other
ingredients of the detergents or by light and do not have any
pronounced substantivity for the textile fibers being treated, so
as not to color them.
To control microorganisms, the washing or cleaning agents may
contain antimicrobial agents. Depending on the antimicrobial
spectrum and the action mechanism, antimicrobial agents are
classified as bacteriostatic agents and bactericides, fungistatic
agents and fungicides, etc. Important substances from these groups
are for example benzalkonium chlorides, alkylaryl sulfonates,
halophenols and phenol mercury acetate. In the context of the
inventive teaching, the expressions "antimicrobial activity" and
"antimicrobial agent" have the usual technical meanings as defined,
for example, by K. H. Wallhau.beta.er in "Praxis der Sterilisation,
Desinfektion-Konservierung Keimidentifizierung-Betriebshygiene"
(5th Edition, Stuttgart/New York: Thieme, 1995), any of the
substances with antimicrobial activity described therein being
usable. Suitable antimicrobials are preferably selected from the
group of the alcohols, amines, aldehydes, antimicrobial acids or
their salts, esters of carboxylic acid, acid amides, phenols,
phenol derivatives, diphenyls, diphenylalkanes, urea derivatives,
oxygen, nitrogen acetals and formals, benzamidines, isothiazolines,
phthalimide derivatives, pyridine derivatives, antimicrobial
surface-active compounds, guanidines, antimicrobial amphoteric
compounds, quinolines, 1,2-dibromo-2,4-dicyanobutane, iodo-2-propyl
butyl carbamate, iodine, iodine-polymer complexes, iodophores,
peroxy compounds, halogen compounds and mixtures of any of the
above.
Consequently, the antimicrobial active substances can be chosen
among ethanol, n-propanol, i-propanol, 1,3-butanediol,
phenoxyethanol, 1,2-propylenelycol, glycerin, undecylenic acid,
benzoic acid, salicylic acid, dihydracetic acid, o-phenylphenol,
N-methylmorpholine-acetonitrile (MMA), 2-benzyl-4-chlorophenol,
2,2'-methylene-bis-(6-bromo-4-chlorophenol),
4,4'-dichloro-2'-hydroxydiphenyl ether (dichlosan),
2,4,4'-trichloro-2'-hydroxydiphenyl ether (trichlosan),
chlorhexidine, N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)-urea,
N,N'-(1,10-decanediyldi-1-pyridinyl-4-ylidene)-bis-(1-octamine)
dihydrochloride,
N,N'-bis-(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraaza-tetradecanediim-
ideamide, glucoprotamines, surface-active antimicrobial quaternary
compounds, guanidines, including the bi- and polyguanidines, such
as for example 1,6-bis(2-ethylhexylbiguanidohexane)
dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-phenyldiguanido-N.sub.5,N.sub.5')hexane
tetrahydrochloride,
1,6-di-(N.sub.1,N.sub.1'-phenyl-N.sub.1,N.sub.1'-methyldiguanido-N.sub.5,-
N.sub.5')hexane dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-.beta.-chlorophenyldiguanido-N.sub.5,
N.sub.5')hexane dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-2,6-dichlorophenyldiguanido-N.sub.5,N.sub.5')hex-
ane dihydrochloride, 1,6-di-[N.sub.1,
N.sub.1'-.beta.-(p-methoxyphenyl)diguanido-N.sub.5,N.sub.5')hexane
dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-.alpha.-methyl-.beta.-phenyldiguanido-N.sub.5,N.-
sub.5')hexane dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-p-nitrophenyldiguanido-N.sub.5,N.sub.5')hexane
dihydrochloride,
.omega.:.omega.-di-(N.sub.1,N.sub.1'-phenyldiguanido-N.sub.5,N.sub.5')di--
n-propyl ether dihydrochloride,
.omega.:.omega.-di-(N.sub.1,N.sub.1'-p-chlorophenyldiguanido-N.sub.5,N.su-
b.5')di-n-propyl ether tetrahydrochloride,
1,6-di-(N.sub.1,N.sub.1'-2,4-dichlorophenyldiguanido-N.sub.5,N.sub.5')hex-
ane tetrahydrochloride,
1,6-di-(N.sub.1,N.sub.1'-p-methylphenyldiguanido-N.sub.5,N.sub.5')hexane
dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-2,4,5-trichlorophenyldiguanido-N.sub.5,N.sub.5')-
hexane tetrahydrochloride, 1,6-di-[N.sub.1,
N.sub.1'-.alpha.-(p-chlorophenyl)ethyldiguanido-N.sub.5,N.sub.5']hexane
dihydrochloride,
.omega.:.omega.-di-(N.sub.1,N.sub.1'-p-chlorophenyldiguanido-N.sub.5,N.su-
b.5')m-xylene dihydrochloride,
1,12-di-(N.sub.1,N.sub.1'-p-chlorophenyldiguanido-N.sub.5,N.sub.5')dodeca-
ne dihydrochloride,
1,10-di-(N.sub.1,N.sub.1'-phenyldiguanido-N.sub.5,N.sub.5')decane
tetrahydrochloride,
1,12-di-(N.sub.1,N.sub.1'-phenyldiguanido-N.sub.5,N.sub.5')dodecane
tetrahydrochloride,
1,6-di-(N.sub.1,N.sub.1'-o-chlorophenyldiguanido-N.sub.5,N.sub.5')hexane
dihydrochloride,
1,6-di-(N.sub.1,N.sub.1'-o-chlorophenyldiguanido-N.sub.5,N.sub.5')hexane
tetrahydrochloride, ethylene-bis-(1-tolylphenyl biguanide),
ethylene-bis-(p-tolylphenylbiguanide),
ethylene-bis-(3,5-dimethylphenylbiguanide),
ethylene-bis-(p-tert-amylphenylbiguanide),
ethylene-bis-(nonylphenylbiguanide),
ethylene-bis-(phenylbiguanide),
ethylene-bis-(N-butylphenylbiguanide),
ethylene-bis-(2,5-diethoxyphenylbiguanide),
ethylene-bis-(2,4-dimethylphenylbiguanide),
ethylene-bis-(o-diphenylbiguanide), ethylene-bis-(mixed
amylnaphthylbiguanide), N-butylethylene-bis-(phenylbiguanide),
trimethylene bis(o-tolylbiguanide),
N-butyltrimethylene-bis-(phenylbiguanide) and the corresponding
salts like acetates, gluconates, hydrochlorides, hydrobromides,
citrates, bisulfites, fluorides, polymaleates, N-coco alkyl
sarcinosates, phosphites, hypophosphites, perfluorooctanoates,
silicates, sorbates, salicylates, maleates, tartrates, fumarates,
ethylenediaminetetraacetates, iminodiacetates, cinnamates,
thiocyanates, arginates, pyromellitates, tetracarboxybutyrates,
benzoates, glutarates, monofluorophosphates, perfluoropropionates
as well as any mixtures thereof. Furthermore, halogenated xylene-
and cresol derivatives are suitable, such as p-chloro-meta-cresol,
p-chloro-meta-xylene, as well as natural antimicrobial active
agents of plant origin (e.g. from spices or aromatics), animal as
well as microbial origin. Preferred antimicrobial agents are
antimicrobial surface-active quaternary compounds, a natural
antimicrobial agent of vegetal origin and/or a natural
antimicrobial agent of animal origin and, most preferably, at least
one natural antimicrobial agent of vegetal origin from the group
comprising caffeine, theobromine and theophylline and essential
oils, such as eugenol, thymol and geraniol, and/or at least one
natural antimicrobial agent of animal origin from the group
comprising enzymes, such as protein from milk, lysozyme and
lactoperoxidase and/or at least one antimicrobial surface-active
quaternary compound containing an ammonium, sulfonium, phosphonium,
iodonium or arsonium group, peroxy compounds and chlorine
compounds. Substances of microbial origin, so-called bacteriozines,
may also be used.
The quaternary ammonium compounds (QUATS) suitable as antimicrobial
agents have the general formula
(R.sup.1)(R.sup.2)(R.sup.3)(R.sup.4)N+X--, in which R.sup.1 to
R.sup.4 may be the same or different and represent C.sub.1-22 alkyl
groups, C.sub.7-28 aralkyl groups or heterocyclic groups, two
or--in the case of an aromatic compound, such as pyridine--even
three groups together with the nitrogen atom forming the
heterocycle, for example a pyridinium or imidazolinium compound,
and X- represents halide ions, sulfate ions, hydroxide ions or
similar anions. In the interests of optimal antimicrobial activity,
at least one of the substituents preferably has a chain length of 8
to 18 and, more preferably, 12 to 16 carbon atoms.
QUATS can be obtained by reacting tertiary amines with alkylating
agents such as, for example, methyl chloride, benzyl chloride,
dimethyl sulfate, dodecyl bromide and also ethylene oxide. The
alkylation of tertiary amines having one long alkyl chain and two
methyl groups is particularly easy. The quaternization of tertiary
amines containing two long chains and one methyl group can also be
carried out under mild conditions using methyl chloride. Amines
containing three long alkyl chains or hydroxy-substituted alkyl
chains lack reactivity and are preferably quaternized with dimethyl
sulfate.
Suitable QUATS are, for example, Benzalkonium chloride
(N-alkyl-N,N-dimethylbenzyl ammonium chloride, CAS No. 8001-54-5),
Benzalkon B (m,p-dichlorobenzyl dimethyl-C1-2-alkyl ammonium
chloride, CAS No. 58390-78-6), Benzoxonium chloride
(benzyldodecyl-bis-(2-hydroxyethyl) ammonium chloride), Cetrimonium
bromide (N-hexadecyl-N,N-trimethyl ammonium bromide, CAS No.
57-09-0), Benzetonium chloride
(N,N-di-methyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy]-eth-
yl]-benzyl ammonium chloride, (CAS No. 121-54-0), dialkyl dimethyl
ammonium chlorides, such as di-n-decyldimethyl ammonium chloride
(CAS No. 7173-51-5-5), didecyldimethyl ammonium bromide (CAS No.
2390-68-3), dioctyl dimethyl ammonium chloride, 1-cetylpyridinium
chloride (CAS No. 123-03-5) and thiazoline iodide (CAS No.
15764-48-1) and mixtures thereof. Particularly preferred QUATS are
the benzalkonium chlorides containing C.sub.8-18 alkyl groups, more
particularly C.sub.12-C.sub.14 alkyl benzyl dimethyl ammonium
chloride.
Benzalkonium halides and/or substituted benzalkonium halides are
commercially available, for example, as Barquat.RTM. from Lonza,
Marquato.RTM. from Mason, Variquat.RTM. from Witco/Sherex and
Hyamine.RTM. from Lonza and as Bardac.RTM. from Lonza. Other
commercially obtainable antimicrobial agents are
N-(3-chloroallyl)-hexaminium chloride, such as Dowicide.RTM. and
Dowicil.RTM. from Dow, benzethonium chloride, such as Hyamine.RTM.
1622 from Rohm & Haas, methyl benzethonium chloride, such as
Hyamine.RTM. 10.times. from Rohm & Haas, cetyl pyridinium
chloride, such as cepacolchloride from Merrell Labs.
The antimicrobial agents are used in quantities of 0.0001% by
weight to 1% by weight, preferably 0.001% by weight to 0.8% by
weight, particularly preferably 0.005% by weight to 0.3% by weight
and most preferably 0.01 to 0.2% by weight.
The inventive detergents or cleaning agents may comprise UV
absorbers that attach to the treated textiles and improve the light
stability of the fibers and/or the light stability of the various
ingredients of the formulation. UV-absorbers are understood to mean
organic compounds, which are able to absorb UV radiation and emit
the resulting energy in the form of longer wavelength radiation,
for example as heat.
Compounds, which possess these desired properties, are for example,
the efficient radiationless deactivating compounds and derivatives
of benzophenone having substituents in position(s) 2- and/or 4.
Also suitable are substituted benzotriazoles, acrylates, which are
phenyl-substituted in position 3 (cinnamic acid derivatives
optionally with cyano groups in position 2), salicylates, organic
Ni complexes, as well as natural substances such as umbelliferone
and the endogenous urocanic acid. The biphenyl and above all the
stilbene derivatives such as for example those described in EP 0
728 749 A and commercially available as Tinosorb.RTM. FD or
Tinosorb.RTM. FR from Ciba, are of particular importance. As UV-B
absorbers can be cited: 3-benzylidenecamphor or
3-benzylidenenorcamphor and its derivatives, for example
3-(4-methylbenzylidene) camphor, as described in the EP 0693471 B1;
4-aminobenzoic acid derivatives, preferably
4-(dimethylamino)benzoic acid, 2-ethylhexyl ester,
4-(dimethylamino)benzoic acid, 2-octyl ester and
4-(dimethylamino)benzoic acid, amyl ester; esters of cinnamic acid,
preferably 4-methoxycinnamic acid, 2-ethylhexyl ester,
4-methoxycinnamic acid, propyl ester, 4-methoxycinnamic acid,
isoamyl ester, 2-cyano-3,3-phenylcinnamic acid, 2-ethylhexyl ester
(octocrylene); esters of salicylic acid, preferably salicylic acid,
2-ethylhexyl ester, salicylic acid, 4-isopropylbenzyl ester,
salicylic acid, homomethyl ester; derivatives of benzophenone,
preferably 2-hydroxy-4-methoxybenzophenone,
2-hydroxy-4-methoxy-4'-methylbenzophenone,
2,2'-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid,
preferably 4-methoxybenzmalonic acid, di-2-ethylhexylester;
triazine derivatives, such as, for example
2,4,6-trianilino-(p-carbo-2'-ethyl-1'-hexyloxy)-1,3,5-triazine and
octyl triazone, as described in EP 0818450 A1 or dioctyl
butamidotriazone (Uvasorb.RTM. HEB); propane-1,3-dione, such as for
example
1-(4-tert.-butylphenyl)-3-(4'-methoxyphenyl)propane-1,3-dione;
ketotricyclo(5.2.1.0) decane derivatives, as described in EP
0694521 B1. Further suitable are 2-phenylbenzimidazole-5-sulfonic
acid and its alkali-, alkaline earth-, ammonium-, alkylammonium-,
alkanolammonium- and glucammonium salts; sulfonic acid derivatives
of benzophenones, preferably
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts;
sulfonic acid derivatives of 3-benzylidenecamphor, as for example
4-(2-oxo-3-bornylidenemethyl)benzene sulfonic acid and
2-methyl-5-(2-oxo-3-bornylidene) sulfonic acid and its salts.
Typical UV-A filters particularly include derivatives of
benzoylmethane, such as, for example
1-(4'-tert.-butylphenyl)-3-(4'-methoxyphenyl)propane-1,3-dione,
4-tert.-butyl-4'-methoxydibenzoylmethane (Parsol 1789),
1-phenyl-3-(4'-isopropylphenyl)-propane-1,3-dione as well as
enamine compounds, as described in the DE 1 971 2033 A1 (BASF).
Naturally, the UV-A and UV-B filters can also be added as mixtures.
Beside the cited soluble materials, insoluble, light protective
pigments, namely finely dispersed, preferably, nano metal oxides or
salts can also be considered for this task. Exemplary suitable
metal oxides are particularly zinc oxide and titanium oxide and
also oxides of iron, zirconium, silicon, manganese, aluminum and
cerium as well as their mixtures. Silicates (talc), barium sulfate
or zinc stearate can be added as salts. The oxides and salts are
already used in the form of pigments for skin care and skin
protecting emulsions and decorative cosmetics. Here, the particles
should have a mean diameter of less than 100 nm, preferably between
5 and 50 nm and especially between 15 and 30 nm. They can be
spherical, however elliptical or other shaped particles can also be
used. The pigments can also be surface treated, i.e. hydrophilized
or hydrophobized. Typical examples are coated titanium dioxides,
such as, for example Titandioxid Z 805 (Degussa) or Eusolex.RTM.
T2000 (Merck); preferably, silicones and particularly preferably
trialkoxy octylsilanes or Simethicones are used as the hydrophobic
coating agents Preferably, micronized zinc oxide is used. Further
suitable UV light protection filters may be found in the review by
P. Finkel in SoFW-Journal, Volume 122 (543), p. 1996.
The UV absorbers are normally used in amounts of 0.01 wt. % to 5
wt. %, preferably from 0.03 wt. % to 1 wt. %.
In general, laundry or cleaning active enzymes are also counted
among the conventional ingredients for detergents and cleaning
agents.
Consequently, detergents or cleaning agents that are characterized
by an above described, inventive protein, protein fragment, fusion
protein or derivative as well as additional further enzymes,
represent preferred embodiments of the present invention. In
particular they include other esterases, proteases, amylases,
cellulases, hemicellulases such as for example .beta.-glucanases,
oxidoreductases such as for example laccases, cutinases and/or
lipases, but also esterases and all other enzymes, which are
described in the prior art for this field of application.
Enzymes like proteases, amylases, lipases or cellulases have been
used for decades as active components in detergents and cleaning
agents. Their respective contribution to the washing or cleaning
performance of the agents in question is, in the case of proteases
the ability to degrade protein-containing stains, in the case of
amylases the degradation of starch-containing stains, and in the
case of lipases the fat cleaving activity.
Cellulases are preferably used in detergents, in particular due to
their contribution to the secondary washing performance of a
detergent and due to their fiber action on textiles, in addition to
their soil-removing, i.e. primary washing and cleaning performance.
The particular hydrolytic products are attacked, dissolved,
emulsified or suspended by the other detergent or cleaning agent
components or are, due to their greater solubility, washed away
with the wash liquor, resulting in synergistic effects between the
enzymes and the other components.
Proteases can exert an effect on natural fibers, in particular on
wool or silk, comparable to the contribution made by cellulase to
the secondary washing performance of a detergent. Due to their
action on the surface structure of such fabrics, they can exert a
smoothing influence on the material and thereby counteract
felting.
Other enzymes extend the cleaning performance of appropriate agents
according to their own specific enzyme performance. Examples of
these include .beta.-glucanases, oxidoreductases such as for
example laccases or pectin-dissolving enzymes, which are used, in
particular, in special detergents.
Enzymes suitable for use in inventive detergents or cleaning agents
are primarily those isolated from microorganisms such as bacteria
or fungi. They are obtained from suitable microorganisms in a
manner known per se by means of fermentation processes.
An inventive protein and/or other proteins present can be
protected, particularly in storage, against deterioration such as,
for example denaturation, decomposition or inactivation, for
example through physical influences, oxidation or proteolytic
cleavage. This is the case for all inventive agents, particularly
detergents and cleaning agents.
One group of stabilizers are reversible protease inhibitors, which
dissociate off when the agent is diluted in the wash liquor.
Benzamidine hydrochloride and leupeptin are established for this
purpose. Frequently, borax, boric acids, boronic acids or salts or
esters thereof are used, including especially the derivatives with
aromatic groups, ortho-substituted, meta-substituted and
para-substituted phenylboronic acids, or salts or esters thereof.
Peptide aldehydes, i.e. oligopeptides with reduced C terminus, that
is those of 2-50 monomers, are employed to reversibly inhibit
detergent and cleaning agent proteases. The peptidic reversible
protease inhibitors include, inter alia, ovomucoid For example,
specific reversible peptide inhibitors of the protease Subtilisin
can be employed in protease-containing agents and corresponding
fusion proteins of protease and inhibitor.
Further enzyme stabilizers are amino alcohols like mono-, di-,
triethanolamine and -propanolamine and their mixtures, aliphatic
carboxylic acids up to C.sub.12, such as succinic acid, other
dicarboxylic acids or salts of the cited acids. End group-capped
fatty amide alkoxylates are disclosed for this purpose in the prior
art. Certain organic acids used as builders are capable of
additionally stabilizing an included enzyme.
Lower aliphatic alcohols, but above all polyols such as, for
example glycerine, ethylene glycol, propylene glycol or sorbitol
are further frequently used enzyme stabilizers. Likewise, calcium
salts are used, such as for example calcium acetate or
calcium-formate, and magnesium salts.
Polyamide oligomers or polymeric compounds-like lignin,
water-soluble vinyl copolymers or cellulose ethers, acrylic
polymers and/or polyamides stabilize enzyme preparations against
physical influences or pH variations. Polymers containing
polyamine-N-oxide act simultaneously as enzyme stabilizers and
color transfer inhibitors. Other polymeric stabilizers are the
linear C.sub.8-C.sub.18 polyoxyalkylenes. Alkyl polyglycosides may
stabilize the enzymatic components of the inventive agent and even
increase their performance. Crosslinked nitrogen-containing
compounds fulfill a dual function as soil release agents and as
enzyme stabilizers. Hydrophobic, non-ionic polymer in a mixture
with other stabilizers acts in a stabilizing manner on a cellulase
such that those or similar components may also be suitable for the
enzyme essential to the invention.
Reducing agents and antioxidants increase the stability of enzymes
against oxidative decomposition. Sulfur-containing reducing agents
are known. Other examples are sodium sulfite and reducing
sugars.
Combinations of stabilizers are also frequently used, for example
of polyols, boric acid and/or borax, the combination of boric acid
or borate, reducing salts and succinic acid or other dicarboxylic
acids or the combination of boric acid or borate with polyols or
polyamino compounds and with reducing salts are disclosed. The
action of peptide-aldehyde stabilizers is increased by combination
with boric acid and/or boric acid derivatives and polyols and still
further increased by the additional use of calcium ions.
Agents containing stabilized enzyme activities are preferred
embodiments of the present invention. Particular preference is
given to those containing enzymes stabilized by more than one of
the illustrated ways.
Since agents of the invention can be provided in any conceivable
form, enzymes or proteins of the invention in any formulations that
are appropriate for addition to the particular agents, represent
respective embodiments of the present invention. Examples thereof
include liquid formulations, solid granules or capsules.
The encapsulated form is a way of protecting the enzymes or other
ingredients against other components such as, for example,
bleaching agents, or of making possible a controlled release.
Depending on their size, said capsules are divided into milli-,
micro- and nanocapsules, microcapsules being particularly preferred
for enzymes. A possible encapsulation method is to encapsulate the
proteins, starting from a mixture of the protein solution with a
solution or suspension of starch or a starch derivative, in this
substance.
In the case of solid agents, the proteins may be used, for example,
in dried, granulated and/or encapsulated form. They can be added
separately, i.e. as one phase, or together with other ingredients
in the same phase, with or without compaction. If
microencapsulated, solid enzymes are used, then the water can be
removed from the aqueous solutions resulting from the process by
means of processes known from the prior art, such as spray-drying,
centrifugation or by transdissolution. The particles obtained in
this manner normally have a particle size between 50 and 200
.mu.m.
Starting from protein recovery carried out according to the prior
art, and preparation in a concentrated aqueous or non-aqueous
solution, suspension or emulsion, but also in gel form or
encapsulated or as dried powder, the inventive enzymes and also the
inventive protein can be added to liquid, gel-like or paste-like
agents of the invention. Such detergents or cleaning agents of the
invention are usually prepared by simply mixing the ingredients,
which may be introduced as solids or as solution into an automated
mixer.
Apart from the primary washing performance, the esterases comprised
in detergents may further fulfill the function of activating, or,
after an appropriate contact time, inactivating other components by
esterolytic cleavage. Comparable regulatory functions are also
possible via the protein of the invention. Another embodiment of
the present invention relates to those agents containing capsules
of esterase-sensitive material, which capsules are hydrolyzed, for
example, by proteins of the invention at the intended time and
release their contents. A comparable effect may also be achieved in
other multi-phase agents.
Agents for the treatment of textile raw materials or for textile
care, which are characterized in that they comprise one of the
above described, inventive proteins, protein fragments, fusion
proteins or derivatives, either alone or in addition to other
ingredients, are a separate subject matter of the invention, in
particular for fibers or textiles with artificial fiber components,
and quite particularly for those containing polyester.
Synthetic fibers, such as for example polyester are also
characterized by a characteristic surface structure. In the long
term, particularly when subjected to a plurality of wash processes,
this can result in undesired effects such as, for example, felting,
"pilling". In order to avoid such effects, either the raw materials
or the finished material, the textile or the fibers are treated
with the inventive agents; this contributes, for example, to
smoothing the surface structure and thereby counteracts
felting.
The inventive agents are preferably used to improve the appearance
and/or the surface structure of already felted (pilled) fibers,
particularly polyester fibers. The inventive enzymes are capable of
cleaving the ester bonds of the felted (pilled) synthetic fibers,
and thereby to reverse (depilling), to prevent from the outset, to
significantly retard and/or even totally stop the felting or
pilling.
In a preferred embodiment, the agent containing an inventive
esterase is designed in such a way that it can be used regularly as
a conditioner, for example by adding it to the washing process,
applying it after washing or independently of the washing. The
desired effect is to obtain a smooth surface structure of the
textile over a long period and/or to prevent and/or reduce damage
to the fabric.
A separate subject matter of the invention is constituted by
processes for the automatic cleaning of textiles or of hard
surfaces, said processes being characterized in that an
above-described, inventive protein, protein fragment, fusion
protein or derivative is active in at least one of the process
steps in an amount of 0.01 .mu.g to 96 g, preferably 0.05 .mu.g to
72 g, particularly preferably 0.1 .mu.g to 48 g and quite
particularly preferably 0.5 .mu.g to 24 g per application.
These processes include both manual as well as automatic processes,
automatic processes being preferred due to their more precise
controllability that concerns for example the added quantities and
contact times.
Processes for the cleaning of textiles are generally characterized
in that various cleaning-active substances are applied to the
material to be cleaned in a plurality of process steps and, after
the contact time, are washed away, or that the material to be
cleaned is treated in any other way with a detergent or a solution
of said agent. The same applies to methods for cleaning any
materials other than textiles, which are classified by the term
hard surfaces. It is possible to add inventive proteins to at least
one of the process steps of all conceivable washing or cleaning
processes; accordingly, these processes then become embodiments of
the present invention.
An individual partial step of such a process for automatic cleaning
of textiles can consist of applying, if desired in addition to
stabilizing compounds, salts or buffer substances, an inventive
enzyme as the single active component. This is a particularly
preferred embodiment of the present invention.
In a further preferred embodiment of such processes, the inventive
enzymes in question are supplied in the context of one of the above
listed formulations for inventive agents, preferably inventive
detergents or cleaning agents.
Preferred embodiments of this subject matter of the invention are
processes for the treatment of textile raw materials or for textile
care, which are characterized in that in at least one of the
process steps one of the above described, inventive proteins,
protein fragments, fusion proteins or derivatives is active,
particularly for textile raw materials, fibers or textiles
containing synthetic components, and quite particularly for those
containing synthetic fibers, particularly polyester.
This can concern processes for example in which materials are
prepared for treating textiles, for example for an anti-pilling
finish or for example processes that add a care component when
cleaning worn textiles. Preferred embodiments concern processes for
the treatment of textile raw materials, fibers or textiles
containing synthetic components, particularly containing synthetic
fibers, preferably polyester.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the cleaning of textiles
or of hard surfaces is a separate subject matter of the
invention.
Preferably, the above listed concentration ranges apply to this
use.
Inventive proteins, particularly corresponding to the above
described characteristics and the above described processes, can be
used for the depilling of textiles. Washing by hand or the manual
removal of blemishes from textiles or from hard surfaces or the use
in connection with an automatic process are exemplary
embodiments.
In a preferred embodiment of this use, the inventive enzymes in
question are supplied in the context of one of the above listed
formulations for inventive agents, preferably inventive detergents
or cleaning agents.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the activation or
deactivation of ingredients of detergents or cleaning agents is a
further embodiment of this subject matter of the invention.
As is known, ingredients of detergents or cleaning agents can be
inactivated by the action of an esterase. A subject matter of the
present invention is to purposely utilize this otherwise rather
undesired effect. It is likewise possible, as described above, that
another component is first activated by esterolytic cleavage, for
example if said component is a hybrid protein of the actual enzyme
and the corresponding inhibitor. Another example of a regulation of
this kind is one in which an active component, in order to protect
or control its activity, has been encapsulated in a material
susceptible to esterolytic attack. Proteins of the invention can
thus be used for inactivation reactions, activation reactions or
release reactions, in particular in multi phase agents.
The use for the controlled and/or retarded release of fragrances,
such as for example perfume oils, is particularly suitable.
All other technical processes, uses and associated agents residing
outside the scope of detergents and cleaning, are summarized,
irrespective of their diversity, in a subject matter of the
invention, in so far as they are characterized by an inventive
protein. This compilation is not to be understood as an exhaustive
listing, but rather assembles the most important presently
recognized application possibilities of the inventive esterases.
Should it transpire that the employment of inventive esterases can
further develop additional technical fields then these are included
in the scope of protection of the present invention.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for biochemical analysis or
for the synthesis of low molecular weight compounds is an
embodiment of this subject matter of the invention.
This use preferably occurs in the context of corresponding agents
or processes. According to the invention, enzymatic analysis is
understood to mean any biochemical analysis that uses specific
enzymes or substrates in order to determine on the one hand the
identity or the concentration of substrates or on the other hand
the identity or activity of enzymes. The fields of application are
all those related to biochemistry, in particular molecular biology
and protein chemistry. This use preferably occurs in the context of
an enzymatic analytical method.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the preparation,
purification or synthesis of natural products or valuable
biological products is a further embodiment of this subject matter
of the invention.
This use preferably occurs in the context of corresponding agents
or processes. Thus, it may be necessary, for example, in the course
of purifying natural products or valuable biological products, to
remove from said products protein contaminations. These, for
example, can be low molecular weight compounds, any cellular
constituents or storage substances or proteins. This may be carried
out both on the laboratory scale and the industrial scale, for
example after the biotechnological production of a valuable
product.
In addition, the use of the inventive proteins in detergents and
cleaning agents can reduce the satin gloss that is observed with
polyester fibers and is regarded as being rather cheap by
consumers. In addition, the formation of glossy areas on synthetic
fibers on washing can be reduced or even essentially prevented.
Glossy areas already formed by strong wear of the fibers, e.g.
rubbing or chafing, are reduced by the use of detergents and
cleaning agents that comprise the inventive enzymes.
Furthermore, the inventive enzymes can be used to reduce or even to
prevent redeposition, i.e. the attachment of soil from the wash
liquor during the washing process.
Moreover, the use of inventive enzymes in detergents and cleaning
agents permits an overall improvement of the cleaning power.
The use of an inventive esterolytic enzyme for the synthesis of
esters or other low molecular weight chemical compounds occurs in
reverse of the reaction naturally catalyzed by it, for example then
when carboxyl groups are esterified or are intended to be bonded
with mono- di- or polyols.
In addition, the esterases, in particular polyesterases, preferably
the para-nitrobenzyl esterases can be employed for chemical
syntheses or purification in a chemical synthesis. As enzymes
generally possess stereoselective catalytic centers, it is possible
to employ the cited enzymes for the separation of racemates or for
stereoselective syntheses.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the treatment of natural
or synthetic raw materials, particularly for surface treatment, is
a further embodiment of this subject matter of the invention.
This use preferably occurs in the context of corresponding agents
or processes. It is required, for example, when certain impurities
have to be eliminated from raw materials. Among these are primarily
understood synthetically obtainable raw materials, such as
polyester compounds, although also substances manufactured
biotechnologically using fermentation, such as for example
antibiotics.
Using polyesterases, in particular p-nitrobenzyl esterases,
preferably obtainable from bacteria of the genus Bacillus, in
processes for the manufacture and/or purification of polyesters, in
particular polyalkylene terephthalates. In the manufacture of
polyalkylene terephthalates, in particular polyethylene
terephthalate (PET), the polymer yield is influenced by a side
reaction that leads to the formation of cyclic oligomers. By means
of the inventive use, it is possible to effect the retro synthesis
of the formed cyclic oligomers and make available the monomers for
further reaction. The products from the side reaction are thus
repressed and a higher yield of desired polymer is achieved.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the production or
treatment of raw materials or intermediates in textile
manufacturing, in particular to remove protective layers on
fabrics, is a further embodiment of this subject matter of the
invention.
This use preferably occurs in the context of corresponding agents
or processes. An example for the production or treatment of raw
materials or intermediates in textile manufacturing is the
finishing of synthetic fibers. Enzymatic processes or uses are
superior to comparable chemical processes, particularly in regard
to their environmental impact.
In a preferred embodiment, inventive proteins are used to remove
protective layers of textiles, in particular intermediates or
materials, or to smooth their surface, prior to further treatment
in a subsequent processing step.
The use of one of the above described, inventive protein, protein
fragment, fusion protein or derivative for the treatment of textile
raw materials or for textile care, in particular for the treatment
of synthetic fibers, particularly polyesters, or mixed textiles
containing synthetic fibers, is a further embodiment of this
subject matter of the invention.
This use preferably occurs in the context of corresponding agents
or processes. In accordance with the above statements, the textile
raw materials in question are freed from impurities by the
esterases; furthermore the surface smoothing and care properties of
the esterolytic enzyme benefit a material consisting at least
partially of protein. For this reason the use for caring for the
materials in question is also included. Consequently, in particular
the surface treatment of synthetic fibers, particularly polyesters,
or of mixed textiles comprising synthetic fibers, is claimed. This
applies both to the preparation of such textiles and also to the
care during usage, for example in connection with the cleaning of
textiles (see above).
In a further embodiment of this subject matter of the invention,
the above described, inventive proteins are employed for cosmetic
purposes.
Consequently, are claimed cosmetics with one of the above described
inventive protein, protein fragment, fusion protein or derivative,
or cosmetic processes involving one of the above described
inventive protein, protein fragment, fusion protein or derivative,
or the use of one of the above described inventive protein, protein
fragment, fusion protein or derivative for cosmetic purposes,
particularly in the context of appropriate processes or in
appropriate agents.
Accordingly, the use of esterolytic enzymes of this kind for
cosmetic purposes, in particular in appropriate agents such as, for
example, shampoos, soaps or washing lotions or in care compositions
provided, for example, in the form of creams, is also included in
this subject matter of the invention. The use in a peeling
medicament and its manufacture is also included in this claim.
A further subject of the invention is the use of inventive
esterases for applications in the manufacture of medicaments,
particularly the manufacture of antibiotics. During synthesis,
carboxyl groups in substances are often protected by a
para-nitrobenzyl protective group. It is therefore preferred to
utilize the inventive esterases in chemical syntheses to cleave the
protecting group, particularly in the manufacture of medicaments,
particularly the manufacture of antibiotics. Preferably, this
occurs in aqueous media at temperatures between 25 and 40.degree.
C.
A further subject of the invention concerns articles of polyester
that result from the inventive use or the application of the
inventive process and/or are produced and/or treated with one of
the abovementioned agents, wherein the article of polyester
possesses an improved handling and feel, depilling characteristics
or protection against pilling.
For the cited inventive uses, those esterases are particularly
preferred, which according to the methods 2.4 cited in the examples
exhibit a specific activity towards the substrate
bis-(p-methylbenzoic acid) ester of ethylene glycol of 0.1 to 30,
preferably 0.6 to 20, particularly 0.7 to 15, quite particularly
preferably 0.9 to 10, even more strongly preferably 1 to 5,
particularly 1.1 to 4, quite particularly preferably 1.5 to 3
(.mu.mol liberated acid)/(min*mg enzyme). These esterases have
proved to be particularly advantageous for use in the inventive
agents or uses and processes.
Furthermore, those esterases are particularly preferred which are
homologous to the protein sequences listed under Seq. ID Nr. 12 to
at least 50%, at least 55%, particularly at least 60%, preferably
at least 65%, particularly preferably at least 70%, advantageously
at least 75%, quite particularly preferably at least 80%,
particularly preferably at least 85%, at least 90%, at least 95%,
at least 99%, especially 100%.
EXAMPLES
1. Sequences of the Inventive Esterases
TABLE-US-00001 TABLE 1 Classification of the sequence ID numbers
SEQ Databank ID Protein/DNA/ entry Esterase/ No. Organism RNA no.
NCBI Lipase 1 Bacillus subtilis Protein -- PNBE 2 Bacillus
licheniformis Protein -- PNBE 3 Bacillus subtilis DNA/RNA -- PNBE 4
Bacillus licheniformis DNA/RNA PNBE 5 Bacillus subtilis Preprotein
PNBE 6 Bacillus licheniformis Preprotein PNBE 7 Bacillus subtilis
DNA/RNA PNBE 8 Bacillus subtilis DNA/RNA PNBE 9 Artificial Signal
peptide PNBE 10 Artificial DNA/RNA PNBE 11 Bacillus subtilis
Protein gi 1762126 PNBE 12 Bacillus licheniformis Protein gi PNBE
DSM13 52346943 13 Bacillus subtilis Protein gi 7546321 PNBE 14
Bacillus subtilis Protein gi 468046 PNBE 15 Bacillus subtilis
Protein gi PNBE 17621265 16 Bacillus subtilis Protein gi 1495277
PNBE 17 Bacillus subtilis Protein gi 1945688 PNBE 18 Bacillus
subtilis Protein gi 2635952 PNBE 19 Bacillus licheniformis Protein
gi PNBE ATCC 14580 52002286 20 Staphylococcus Protein gi PNBE
epidermis ATCC 27316488 12228 21 Staphylococcus Protein gi PNBE
aureus 57286454 22 Streptomyces Protein gi PNBE avermitilis MA-4860
29611030 23 Caulobacterium Protein gi PNBE crescentus CB15 13422044
24 Clostridium Protein gi PNBE acetobutylicum 14994366 ATCC824 25
Artificial Protein gi 7546320 PNBE 26 Burkholderia Protein gi
Lipase cepacia 67464317 27 Bacillus licheniformis DNA PNBE
DSM13
2. Determination of the Enzyme Activities
2.1 Determination of Esterase Activity with Para-Nitrophenyl
Esters
The detection of the esterase activity was determined using
standard methods with para-nitrophenyl acetate (pNPA). For this a
400 mM parent solution was mixed with DMSO. 980 .mu.L of a 50 mM
tris-HCl-buffer (pH 8.0) were mixed with 10 .mu.L para-nitrophenyl
acetate dissolved in DMSO. The reaction was carried out--if not
otherwise described--at room temperature and started by adding 10
.mu.L of enzyme solution. The increase in absorption at 410 nm was
then measured (UV MC2, SAFAS, Monaco). The enzyme activity was
calculated with a molar extinction coefficient of 17100 [M-1 cm-1],
wherein
1 unit was defined as the quantity of enzyme that released 1
.mu.mol p-nitrophenol per minute.
2.2 Determination of Esterase Activity with Phenol Red
For the biochemical purification of dialkyl phthalate-hydrolases,
esterolytic activity determinations were used against diethyl
terephthalate (DET) as the substrate with a photometric measurement
method that showed the acidification of the test system by means of
pH indicators. The basic principle of this test system is the same
proton affinity of the added buffer (EPPS-P.sub.uffer;
PK.sub.a.dbd.8.0) and of the added pH indicator (phenol red;
pK.sub.a.dbd.8.0), which enable a linear trend of the decrease in
absorption at 560 nm. To calculate the activity, the following
formula 2.1 and 2.2 were used with Q=buffer factor;
cb.sub.uffer.dbd.final concentration of buffer;
c.sub.indicator.dbd.final concentration of indicator;
.DELTA..epsilon..sub.560nm: difference of the deprotonated form and
protonated form of phenol red; A: activity of the esterase; dE/dt:
extinction decrease over time; V.sub.reaction: reaction volume:
.DELTA..times. .mu..times..times. dd ##EQU00001##
The enzyme activity was calculated using an empirically determined
molar extinction coefficient of 58000 [M.sup.-1cm.sup.-1] for the
deprotonated and 100 [M.sup.-1cm.sup.-1] for the protonated form,
wherein 1 unit is defined as the quantity of enzyme that releases 1
.mu.mol acid per minute.
2.3 Determination of the Esterase Activity by Titration
The enzyme activities of the esterases were measured with the
titration apparatus 702 SAT (METROHM Ltd., Switzerland) for various
substrates of the phthalate family (FIG. 4). The phthalates were
dissolved in diethyl ether with 0.5 g Triton X100. The diethyl
ether was then removed under a stream of nitrogen. 50 ml buffer (2
mM Tris/HCl, pH 9) was added, homogenized by ultrasound and used in
different final concentrations (0.3 mM to 100 mM) in a total
reaction volume of 2 mL (including enzyme). The reaction buffer was
pre-incubated at 30.degree. C. for 10 minutes. After addition of
the enzyme, the NaOH consumption (concentration determination by
triple titration with oxalic acid) at a reaction temperature of
30.degree. C. was plotted. One unit is defined as the enzyme
activity that releases 1 .mu.mol acid per minute.
2.4 Determination of the Esterase Activity by Hydrolysis of
Bis-(P-Methylbenzoic Acid) Ester of Ethylene Glycol
2.4.1 Synthesis of Bis-(P-Methylbenzoic Acid) Ester of Ethylene
Glycol
Bis-(p-methylbenzoic acid) ester of ethylene glycol was synthesized
by the esterification of 4-methylbenzoyl chloride and ethylene
glycol (see FIG. 1). Ethylene glycol and pyridine were placed in a
round bottomed flask equipped with a reflux column and
4-methylbenzoyl chloride was added drop wise under ice cooling. The
mixture was allowed to stand for 16 hours and then the resulting
solid ester was filtered off and recrystallized in 70% ethanol 30%
water. The resulting solid was then washed with 70% ethanol and
lyophilised for 24 hours.
2.4.2 Determination of the Enzymatic Activity
The cleavage of the bis-(p-methylbenzoic acid) ester of ethylene
glycol catalyzed by the inventive enzymes affords 2 equivalents of
4-methylbenzoic acid and ethylene glycol (see FIG. 2).
For the preparation of the enzyme assay, the desired amount of
substrate was mixed with 0.5 g Triton X100 and dissolved with
heating in 50 mL ethanol (99%, denatured). Once all the substrate
was dissolved, the solution was added to 50 mL buffer (2 mM
tris/HCl buffer; pH 9.0) with intense stirring by Ultra-Turrax.
This solution was then stirred at room temperature for at least 12
hours in order to remove a large part of the added ethanol. The
thus-prepared substrate solution was immediately titrated. The
reaction buffer was pre-incubated at 30.degree. C. for 10
minutes.
Different final concentrations (0.67 mM to 50 mM) were used in a
total reaction volume of 2 mL including enzyme. After addition of
the enzyme, the NaOH consumption (concentration determination by
triple titration with oxalic acid) with the titration apparatus 702
SAT (METROHM Ltd., Switzerland)) at a reaction temperature of
30.degree. C. was plotted. One unit is defined as the enzyme
activity that releases 1 .mu.mol acid per minute.
In this and the subsequent examples, the following abbreviations
are used for the tested esterases: enzyme according to Seq ID No.
12 (pNB-Est13), enzyme according to Seq ID No. 2 (pNB-Est19),
enzyme according to Seq ID No. 1 (pNB-Est17). The data obtained for
the cited esterases in Table 2 were determined by titrimetric
measurements and represent mean values from: three independent
measurements with a relative standard deviation of less than
5%.
TABLE-US-00002 TABLE 2 Kinetic data of the hydrolysis of the
bis-(p-methylbenzoic acid) ester of ethylene glycol by
para-nitrobenzyl esterases pNB-Est17, pNB-Est19 and pNB-Est13
bis-(p- methylbenzoic acid) ester of v.sub.max Specific ethylene
[.mu.mol/ k.sub.M k.sub.cat k.sub.cat/k.sub.M Activity glycol min]
[mM] [1/s] [1/mM * s] [.mu.mol/min * mg] Enzyme acc. 0.642 9.83
5177 527 1.60 Seq ID No. 1 (pNB-Est17) Enzyme acc. 0.385 3.59 3838
1070 1.19 Seq ID No. 2 (pNB-Est19) Enzyme acc. 1.801 11.21 9358 835
2.87 Seq ID No. 12 (pNB-Est13)
3. Investigation of the Substrate Specificity to Para-Nitrophenyl
Esters and Triglycerides of Different Chain Lengths
For the determination of chain length specificities of the
investigated esterases, p-nitrophenyl esters with different chain
lengths were used as the substrate for the spectroscopic
measurements of activity. To the p-nitrophenyl esters dissolved in
10 mL isopropanol were added 90 mL phosphate buffer (pH 8, disodium
hydrogen phosphate and potassium dihydrogen phosphate). The final
concentration of the substrate was 0.8 mM. Each 990 .mu.L
buffer-substrate mixture was mixed with 10 .mu.L of the enzyme
sample and incubated at room temperature for one minute. The
increase in absorption at 410 nm was then measured (UV MC.sup.2,
SAFAS, Monaco). The enzyme activity was calculated using a molar
extinction coefficient of 17100 [M.sup.-1cm.sup.-1], wherein 1 unit
was defined as the quantity of enzyme that released 1 .mu.mol
p-nitrophenol per minute.
The data in Table 3 represent mean values from three independent
measurements with a relative standard deviation of less than
10%.
TABLE-US-00003 TABLE 3 Substrate specificities of pNB-Est13.
pNB-Est17 and pNB-Est19 with different chain lengths of the
para-nitrophenyl esters Specific Activity [U/mg] Substrate
pNB-Est13 pNB-EST17 pNB-Est19 p-NP-butyrate 22.4 196.3 234.8
p-NP-caproate 50.5 102.7 249.6 p-NP-caprylate 13.0 73.2 83.6
p-NP-caprate 4.4 10.2 3.9 p-NP-laurate 1.3 3.4 4.0 p-NP-myristate
<1 <1 1.3 p-NP-palmitate <1 <1 <1 p-NP-stearate
<1 <1 <1
4. Analytical Detection of the Hydrolysis Products of Various
Dialkyl Phthalates after Incubation with the Para-Nitrobenzyl
Esterases pNB-Est17, pNB-Est19 and pNB-Est13
The esterolytic activity of the pNB esterases towards different
dialkyl phthalates was verified by detecting the products of
cleavage. The detection involved both thin layer chromatography and
gas chromatography coupled to mass spectroscopy (GC/MS). The
RF-values of the starting materials and reaction products
determined by thin layer chromatography are shown in Table 3. The
detection of one or two new spots indicates hydrolysis. Each of the
blind samples exhibited no additional spots.
A mixture of 90% dichloromethane/10% methanol was used as the
eluent in a presaturated TLC chamber; the separation of the
substances was carried out with silica gel 60 F254 coated plates;
detection of the individual spots was made under UV light;
substrate concentration in the mixture was 6.67 mM, incubation time
2 h at room temperature.
TABLE-US-00004 TABLE 4 Characteristic RF values of the starting
materials and products determined by thin layer chromatographic
analyses of the reaction mixtures containing pNB esterases.
Substrate RF Substrate RF Product 1 RF Product 2 Dimethyl 0.78 0.18
terephthalate Diethyl 0.75 0.21 terephthalate Dimethyl 0.78 0.23
isophthalate Dimethyl 0.70 0.07 phthalate Diethyl phthalate 0.73
0.13 Dibutyl phthalate 0.74 0.13 Phenyl benzoate 0.76 0.45 0.25
Peak assignments of the mass peaks from GC/MS of the cleavage
products of the reaction mixtures were carried out by comparison
with the WILEY databank and confirmed the hydrolysis that had
already been observed by thin layer chromatographic analyses. All
observed reactions are schematically illustrated in FIG. 4. Each
enzymatic reaction was measured against a blank sample that
consisted of the reaction mixture without enzyme or the reaction
mixture without substrate. It was shown that for all investigated
phthalates and terephthalates, only one of the two possible ester
functional groups is hydrolyzed. Free phthalic acid or terephthalic
acid or isophthalic acid was not detected in any reaction mixture.
In each case the reaction products are the monoester of the
starting compound and the relevant alcohol. The hydrolysis of
phenyl benzoate was investigated as a substrate that did not belong
to the phthalates. Phenol and benzoic acid were detected as the
hydrolysis products.
5. Determination of v.sub.max, k.sub.M, k.sub.cat and
k.sub.cat/k.sub.M of the para-nitrobenzyl Esterases pNB-Est17,
pNB-Est19 and pNB-Est13 Towards Different Phthalates and
Terephthalates
Titrimetric measurements were carried out to determine the kinetic
constants v.sub.max, k.sub.M, k.sub.cat and k.sub.cat/k.sub.M of
the three para-nitrobenzyl esterases pNB-Est17, pNB-Est19 and
pNB-Est13 towards different phthalates and terephthalates. The
enzyme activities were determined for different substrate
concentrations and evaluated according to MICHAELIS-MENTEN (see
FIGS. 5 to 7). The measured k.sub.M- and v.sub.max values and the
added enzyme concentrations were used to calculate the kinetic
constants (k.sub.cat and k.sub.2) and the catalytic efficiency
(k.sub.cat/k.sub.M)--under the assumption that the added enzyme
concentration was the same as the concentration of the active
enzyme. The resulting values are each summarized in the tables.
The data in FIGS. 5 to 7 and Tables 5 to 7 represent mean values
from three independent titrimetric measurements with a relative
standard deviation of less than 5%. The calculation of k.sub.cat
and k.sub.cat/k.sub.M was based on the assumption that the added
enzyme concentration was the same as the concentration of the
active enzyme.
The monoester of the starting compound with the relevant alcohol
could be detected for all three investigated para-nitrobenzyl
esterases. In no case could the free phthalic acid or terephthalic
acid or isophthalic acid be detected.
Hydrolysis of the Different Isomers (Ortho, Meta, Para) of Dimethyl
Phthalates.
TABLE-US-00005 TABLE 5 k.sub.M and V.sub.max values and k.sub.cat
and k.sub.cat/k.sub.M of the hydrolysis of ortho, meta, para
positional isomers of dimethyl phthalate, DMP = dimethyl phthalate;
DMIP = dimethyl isophthalate; DMT = dimethyl terephthalate by
pNB-Est17, pNB-Est19 and pNB-Est13. pNB-Est17 pNB-Est19 pNB-Est13
Substrat DMP v.sub.Max [U/mg] 14.09 15.76 9.11 k.sub.M [mM] 27.70
20.35 8.68 k.sub.cat [1/s] 45672 50970 29735 k.sub.cat/k.sub.M
[1/mM * s] 1649 2505 3427 Substrat DMIP v.sub.Max [U/mg] 11.99 8.55
27.67 k.sub.M [mM] 3.67 2.44 2.93 k.sub.cat [1/s] 38880 27637 90267
k.sub.cat/k.sub.M [1/mM * s] 10594 11327 30808 Substrat DMT
v.sub.Max [U/mg] 18.52 15.69 4.53 k.sub.M [mM] 5.99 13.56 6.87
k.sub.cat [1/s] 60028 50718 14784 k.sub.cat/k.sub.M [1/mM * s]
10021 3741 2152 Substrat = substrate
Hydrolysis of (Ortho) Phthalates with Different Chain Lengths
TABLE-US-00006 TABLE 6 k.sub.M and V.sub.max values and k.sub.cat
and k.sub.cat/k.sub.M of the hydrolysis of ortho phthalates with
different alkyl chain lengths (DMP = dimethyl phthalate: DEP =
diethyl phthalate: DBP = dibutyl phthalate: by pNB-Est17, pNB-Est19
and pNB-Est13. pNB-Est17 pNB-Est19 pNB-Est13 Substrat DMP v.sub.Max
[U/mg] 14.09 15.76 9.11 k.sub.M [mM] 27.70 20.35 8.68 k.sub.cat
[1/s] 45672 50970 29735 k.sub.cat/k.sub.M [1/mM * s] 1649 2505 3427
Substrat DEP v.sub.Max [U/mg] 9.57 7.20 3.21 k.sub.M [mM] 5.20 3.55
4.14 k.sub.cat [1/s] 31065 23265 10468 k.sub.cat/k.sub.M [1/mM * s]
5974 6554 2528 Substrat DBP v.sub.Max [U/mg] 48.03 40.69 2.87
k.sub.M [mM] 1.24 0.70 1.16 k.sub.cat [1/s] 155676 23265 9356
k.sub.cat/k.sub.M [1/mM * s] 125545 187329 8066 Substrat =
substrate
Hydrolysis of (Para) Phthalates with Different Chain Lengths
TABLE-US-00007 TABLE 7 k.sub.M and V.sub.max values and k.sub.cat
and k.sub.cat/k.sub.M of the hydrolysis of dimethyl terephthalate
(DMT) diethyl terephthalate (PET) by pNB-Est17, pNB-Est19 and
pNB-Est13. pNB-Est17 pNB-Est19 pNB-Est13 Substrat DMT v.sub.Max
[U/mg] 18.52 15.69 4.53 k.sub.M [mM] 5.99 13.56 6.87 k.sub.cat
[1/s] 60028 50718 14784 k.sub.cat/k.sub.M [1/mM * s] 10021 3741
2152 Substrat DET v.sub.Max [U/mg] 28.06 30.79 3.57 k.sub.M [mM]
1.06 0.97 1.21 k.sub.cat [1/s] 90998 99411 12574 k.sub.cat/k.sub.M
[1/mM * s] 85847 102486 10392 Substrat = substrate
NOTES TO THE FIGURES
FIG. 1: Synthesis of bis-(p-methylbenzoic acid) ethylene glycol
ester by esterification of 4-methylbenzoic acid and ethylene
glycol
FIG. 2: Scheme of the enzymatic hydrolysis of bis-(p-methylbenzoic
acid) ethylene glycol ester
FIG. 3. Detection of the enzymatic hydrolysis and identification of
the hydrolysis products of bis-(p-methylbenzoic acid) ethylene
glycol ester by para-nitrobenzyl esterase
FIG. 4: Schematic illustration of the enzyme catalyzed reactions of
dimethyl phthalate, diethyl phthalate, dimethyl phthalate, diethyl
phthalate, dibutyl phthalate, dimethyl isophthalate and phenyl
benzoate.
FIG. 5: MICHAELIS-MENTEN kinetics of the para-nitrobenzyl esterases
pNB-Est17 (o), pNB-Est19 (.DELTA.) and pNB-Est13 (.quadrature.)
towards dimethyl phthalate (DMP), dimethyl isophthalate (DMIP) and
dimethyl terephthalate (DMT)
FIG. 6: MICHAELIS-MENTEN kinetics of the para-nitrobenzyl esterases
pNB-Est17 (o), pNB-Est19 (.DELTA.) and pNB-Est13 (.quadrature.)
towards ortho phthalates: dimethyl phthalate (DMP), diethyl
phthalate (DEP) and dibutyl phthalate (DBP).
FIG. 7: MICHAELIS-MENTEN kinetics of the para-nitrobenzyl esterases
pNB-Est17 (o), pNB-Est19 (.DELTA.) and pNB-Est13 (.quadrature.)
towards para phthalates: dimethyl terephthalate (DMT) and diethyl
terephthalate (DET)
SEQUENCE LISTINGS
1
271489PRTBacillus subtilis 1Met Thr His Gln Ile Val Thr Thr Gln Tyr
Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp Lys
Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe Lys
Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala Thr
Ala Tyr Gly Pro Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser Leu
Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu Tyr
Val Asn Val Phe Ala Pro Asp Thr Pro Ser Gln Asn 85 90 95Leu Pro Val
Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105 110Gly
Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu 115 120
125Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu
130 135 140His Leu Ser Ser Phe Asp Glu Ala Tyr Ser Asp Asn Leu Gly
Leu Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val Arg Glu
Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val Thr Val
Phe Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala Leu Leu
Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala Ile Met
Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln Ala Ala
Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230 235
240Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu Leu
245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile
Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr Leu
Pro Ala Glu Pro 275 280 285Glu Lys Ala Ile Ser Glu Gly Ala Ala Ser
Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly Tyr
Leu Phe Phe Thr Pro Asp Ser Asp305 310 315 320Val His Ser Gln Glu
Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro Leu
Ala Glu Lys Ala Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345 350Ser
Gln Ile His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val 355 360
365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg
370 375 380Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe
His Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn Leu Asp
Gly Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp Glu Val
Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Ile Thr Phe
Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn Trp Pro
Thr Tyr His Glu Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu Asp Ser
Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470 475
480Gln Lys Leu Phe Pro Ser Lys Gly Glu 4852489PRTBacillus
licheniformis 2Met Thr His Gln Ile Val Thr Thr Gln Tyr Gly Lys Val
Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp Lys Gly Ile Pro
Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe Lys Ala Pro Glu
Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala Thr Ala Tyr Gly
Pro Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser Leu Ser Tyr Thr
Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu Tyr Val Asn Val
Phe Ala Pro Asp Thr Pro Ser Gln Asn 85 90 95Leu Pro Val Met Val Trp
Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105 110Gly Ser Glu Pro
Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu 115 120 125Val Ile
Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu 130 135
140His Leu Ser Ser Phe Asp Glu Ala Tyr Ser Asp Asn Leu Gly Leu
Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val Arg Glu Asn
Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val Thr Val Phe
Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala Leu Leu Ala
Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala Ile Met Glu
Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln Ala Ala Ser
Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230 235 240Glu
Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu Leu 245 250
255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln
260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Ala
Glu Pro 275 280 285Glu Lys Ala Ile Ser Glu Gly Ala Ala Ser Gly Ile
Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly Tyr Leu Phe
Phe Thr Pro Asp Ser Asp305 310 315 320Val His Ser Gln Glu Thr Leu
Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro Leu Ala Glu
Lys Ala Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345 350Ser Gln Ile
His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val 355 360 365Ala
Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg 370 375
380Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe His
Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn Leu Asp Gly
Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp Glu Val Lys
Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Ile Thr Phe Ala
Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn Trp Pro Thr
Tyr His Glu Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu Asp Ser Glu
Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470 475 480Gln
Lys Leu Phe Pro Ser Lys Gly Glu 48531470DNABacillus subtilis
3atgactcatc aaatagtaac gactcaatac ggcaaagtaa aaggcacaac ggaaaacggc
60gtacataagt ggaaaggcat cccctatgcc aagccgcctg tcggacaatg gcgttttaaa
120gcacctgagc cgcctgaagt gtgggaagat gtccttgatg ccacagcgta
cggccctatt 180tgcccgcagc cgtctgattt gctctcactg tcgtatacag
agctgccccg ccagtccgag 240gattgcttgt atgtcaatgt atttgcgcct
gacaccccaa gtcaaaatct tcctgtcatg 300gtgtggattc acggaggcgc
tttttatctt ggagcgggca gtgagccatt gtatgacgga 360tcaaaacttg
cggcacaggg agaagtcatt gtcgttacat tgaactatcg gctggggccg
420tttggctttt tgcacttgtc ttcgtttgat gaggcgtatt ccgataacct
tgggctttta 480gaccaagccg ccgcactgaa atgggtgcgg gagaatattt
cagcgtttgg cggtgatccc 540gataacgtaa cagtatttgg agaatccgcc
ggcgggatga gcattgccgc gcttctcgct 600atgcctgcgg caaaaggcct
gttccagaaa gcaatcatgg aaagcggcgc ttctcgaacg 660atgacgaaag
aacaagcggc gagcacctcg gcagcctttt tacaggtcct tgggattaac
720gagggccaat tggataaatt gcatacggtt tctgcagaag atttgctaaa
agcggctgat 780cagcttcgga ttgcagaaaa agaaaatatc tttcagctgt
tcttccagcc cgcccttgat 840ccaaaaacgc tgcctgctga accagaaaaa
gcgatctcag aaggggctgc ttccggcatt 900cctctattga ttggaacaac
ccgtgatgaa ggatatttat ttttcacccc ggattcagac 960gttcattctc
aggaaacgct tgatgcagca ctcgagtatt tactagggaa gccgctggca
1020gagaaagctg ccgatttgta tccgcgttct ctggaaagcc aaattcatat
gatgactgat 1080ttattatttt ggcgccctgc cgtcgcctat gcatccgcac
agtctcatta cgcccctgtc 1140tggatgtaca ggttcgattg gcacccgaag
aagccgccgt acaataaagc gtttcacgca 1200ttagagcttc cttttgtctt
tggaaatctg gacggattgg aacgaatggc aaaagcggag 1260attacggatg
aggtgaaaca gctttctcac acgatacaat cagcgtggat cacgttcgct
1320aaaacaggaa acccaagcac cgaagctgtg aattggccga cgtatcatga
agaaacgaga 1380gagacgctga ttttagattc agagattacg atcgaaaacg
atcccgaatc tgaaaaaagg 1440cagaagctat tcccttcaaa aggagaataa
147041470DNABacillus licheniformis 4atgtctcata aaacagtaac
aactcaatac ggcaaagtaa aaggcacaac agaaaacggc 60gtacataaat ggaaaggcat
cccctatgcc aaacctcctg tcgggccatt gcgttttaaa 120gcaccggaac
ctcctgaagc gtgggagaac gaactggacg caacagcata cggctctatt
180tgcccgcagc cgtctgattt gctgtcactt tcgtatactg agctgccccg
ccagtctgag 240gattgcttgt atatcaatgt atttgcgcct gatactccaa
gtcaaaacct gcctgtcatg 300gtatggattc acggcggcgc tttttatctt
ggagcgggca gtgagccatt atatgatggg 360tcaagacttg cggcgcaggg
agaagtcatt gtcgttacac tgaattatcg tctggggccg 420tttggatttt
tacatttgtc ttcgtttgaa gagacgtatt ccgataacct tgggcttttg
480gaccaagccg ccgcactgaa atgggtgcga gacaatatct cagcatttgg
cggtgatccg 540gataacgtaa cagtatttgg agaatcagca ggcggcatga
gcattgccgc gctgctcgca 600atgcctgcgg caaaaggcct gttccagaaa
gcaatcatgg aaagtggcgc ttccagaacg 660atgacaaaag aaaaagcggc
tagcaccgcg gcagcctttt tagaggtcct tgggattgac 720gagagccaat
tggacaggtt gcatactgta tctgcggaag atttgcttaa agcggccgat
780cagcttcgga aagcagaaaa tgaaaatctc tttcagctgt tcttccagcc
cgcccttgat 840ccgaaaacgc tgcctgctga accagaaaaa gcgatcgcag
agggtgctgc tgccggcatt 900ccgctgttaa tcggaacaaa ccgcgatgaa
ggatatttat ttttcacccc ggactcagac 960gttcattctc aggaaacgtt
tgatgccgcg cttgtgtatt tattagggca gccgctggca 1020gagaaagccg
ccgatctgta tccgcgttcg ctggaaagcc aaattcatat gatgactgat
1080ttgttatttt ggcgcccggc cgtcgcctgt gcctccgcac agtcccatta
cgcgcctgtc 1140tggatgtacc gattcgattg gcactctgat aagccgccgt
ataataaagc gtttcacgca 1200ttagagcttc cttttgtttt cggaaatctg
gacgggttag aacggatggc aaaagcagag 1260attacggatg aagtgaaaca
gctctctcac accatacaat cagcatggat cacgttcgcc 1320aaaacaggga
acccaagcac tgaagatgta aaatggccgg cgtatcatga ggaaacaaga
1380gagacgctga ttttaaattc agagattgcg attgaaaacg accctgaagc
tgaaaaaagg 1440cagaaactat tcccttcaca aggagaataa 14705518PRTBacillus
subtilis 5Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala
Phe Met1 5 10 15Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala
Met Thr His 20 25 30Gln Ile Val Thr Thr Gln Tyr Gly Lys Val Lys Gly
Thr Thr Glu Asn 35 40 45Gly Val His Lys Trp Lys Gly Ile Pro Tyr Ala
Lys Pro Pro Val Gly 50 55 60Gln Trp Arg Phe Lys Ala Pro Glu Pro Pro
Glu Val Trp Glu Asp Val65 70 75 80Leu Asp Ala Thr Ala Tyr Gly Pro
Ile Cys Pro Gln Pro Ser Asp Leu 85 90 95Leu Ser Leu Ser Tyr Thr Glu
Leu Pro Arg Gln Ser Glu Asp Cys Leu 100 105 110Tyr Val Asn Val Phe
Ala Pro Asp Thr Pro Ser Gln Asn Leu Pro Val 115 120 125Met Val Trp
Ile His Gly Gly Ala Phe Tyr Leu Gly Ala Gly Ser Glu 130 135 140Pro
Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu Val Ile Val145 150
155 160Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu His Leu
Ser 165 170 175Ser Phe Asp Glu Ala Tyr Ser Asp Asn Leu Gly Leu Leu
Asp Gln Ala 180 185 190Ala Ala Leu Lys Trp Val Arg Glu Asn Ile Ser
Ala Phe Gly Gly Asp 195 200 205Pro Asp Asn Val Thr Val Phe Gly Glu
Ser Ala Gly Gly Met Ser Ile 210 215 220Ala Ala Leu Leu Ala Met Pro
Ala Ala Lys Gly Leu Phe Gln Lys Ala225 230 235 240Ile Met Glu Ser
Gly Ala Ser Arg Thr Met Thr Lys Glu Gln Ala Ala 245 250 255Ser Thr
Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn Glu Gly Gln 260 265
270Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu Leu Lys Ala Ala
275 280 285Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln Leu
Phe Phe 290 295 300Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Ala Glu
Pro Glu Lys Ala305 310 315 320Ile Ser Glu Gly Ala Ala Ser Gly Ile
Pro Leu Leu Ile Gly Thr Thr 325 330 335Arg Asp Glu Gly Tyr Leu Phe
Phe Thr Pro Asp Ser Asp Val His Ser 340 345 350Gln Glu Thr Leu Asp
Ala Ala Leu Glu Tyr Leu Leu Gly Lys Pro Leu 355 360 365Ala Glu Lys
Ala Ala Asp Leu Tyr Pro Arg Ser Leu Glu Ser Gln Ile 370 375 380His
Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val Ala Tyr Ala385 390
395 400Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg Phe Asp
Trp 405 410 415His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe His Ala
Leu Glu Leu 420 425 430Pro Phe Val Phe Gly Asn Leu Asp Gly Leu Glu
Arg Met Ala Lys Ala 435 440 445Glu Ile Thr Asp Glu Val Lys Gln Leu
Ser His Thr Ile Gln Ser Ala 450 455 460Trp Ile Thr Phe Ala Lys Thr
Gly Asn Pro Ser Thr Glu Ala Val Asn465 470 475 480Trp Pro Thr Tyr
His Glu Glu Thr Arg Glu Thr Leu Ile Leu Asp Ser 485 490 495Glu Ile
Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg Gln Lys Leu 500 505
510Phe Pro Ser Lys Gly Glu 5156518PRTBacillus licheniformis 6Met
Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met1 5 10
15Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Met Thr His
20 25 30Gln Ile Val Thr Thr Gln Tyr Gly Lys Val Lys Gly Thr Thr Glu
Asn 35 40 45Gly Val His Lys Trp Lys Gly Ile Pro Tyr Ala Lys Pro Pro
Val Gly 50 55 60Gln Trp Arg Phe Lys Ala Pro Glu Pro Pro Glu Val Trp
Glu Asp Val65 70 75 80Leu Asp Ala Thr Ala Tyr Gly Pro Ile Cys Pro
Gln Pro Ser Asp Leu 85 90 95Leu Ser Leu Ser Tyr Thr Glu Leu Pro Arg
Gln Ser Glu Asp Cys Leu 100 105 110Tyr Val Asn Val Phe Ala Pro Asp
Thr Pro Ser Gln Asn Leu Pro Val 115 120 125Met Val Trp Ile His Gly
Gly Ala Phe Tyr Leu Gly Ala Gly Ser Glu 130 135 140Pro Leu Tyr Asp
Gly Ser Lys Leu Ala Ala Gln Gly Glu Val Ile Val145 150 155 160Val
Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu His Leu Ser 165 170
175Ser Phe Asp Glu Ala Tyr Ser Asp Asn Leu Gly Leu Leu Asp Gln Ala
180 185 190Ala Ala Leu Lys Trp Val Arg Glu Asn Ile Ser Ala Phe Gly
Gly Asp 195 200 205Pro Asp Asn Val Thr Val Phe Gly Glu Ser Ala Gly
Gly Met Ser Ile 210 215 220Ala Ala Leu Leu Ala Met Pro Ala Ala Lys
Gly Leu Phe Gln Lys Ala225 230 235 240Ile Met Glu Ser Gly Ala Ser
Arg Thr Met Thr Lys Glu Gln Ala Ala 245 250 255Ser Thr Ser Ala Ala
Phe Leu Gln Val Leu Gly Ile Asn Glu Gly Gln 260 265 270Leu Asp Lys
Leu His Thr Val Ser Ala Glu Asp Leu Leu Lys Ala Ala 275 280 285Asp
Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln Leu Phe Phe 290 295
300Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Ala Glu Pro Glu Lys
Ala305 310 315 320Ile Ser Glu Gly Ala Ala Ser Gly Ile Pro Leu Leu
Ile Gly Thr Thr 325 330 335Arg Asp Glu Gly Tyr Leu Phe Phe Thr Pro
Asp Ser Asp Val His Ser 340 345 350Gln Glu Thr Leu Asp Ala Ala Leu
Glu Tyr Leu Leu Gly Lys Pro Leu 355 360 365Ala Glu Lys Ala Ala Asp
Leu Tyr Pro Arg Ser Leu Glu Ser Gln Ile 370 375 380His Met Met Thr
Asp Leu Leu Phe Trp Arg Pro Ala Val Ala Tyr Ala385 390 395 400Ser
Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg Phe Asp Trp 405 410
415His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe His Ala Leu Glu Leu
420 425 430Pro Phe Val Phe Gly Asn Leu Asp Gly Leu Glu Arg Met Ala
Lys Ala 435 440 445Glu Ile Thr Asp Glu Val Lys Gln Leu Ser His Thr
Ile Gln Ser Ala 450 455 460Trp Ile Thr Phe Ala Lys Thr Gly Asn
Pro
Ser Thr Glu Ala Val Asn465 470 475 480Trp Pro Thr Tyr His Glu Glu
Thr Arg Glu Thr Leu Ile Leu Asp Ser 485 490 495Glu Ile Thr Ile Glu
Asn Asp Pro Glu Ser Glu Lys Arg Gln Lys Leu 500 505 510Phe Pro Ser
Lys Gly Glu 51571557DNABacillus subtilis 7atgatgagga aaaagagttt
ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60atggcattca gcgattccgc
ttctgctatg actcatcaaa tagtaacgac tcaatacggc 120aaagtaaaag
gcacaacgga aaacggcgta cataagtgga aaggcatccc ctatgccaag
180ccgcctgtcg gacaatggcg ttttaaagca cctgagccgc ctgaagtgtg
ggaagatgtc 240cttgatgcca cagcgtacgg ccctatttgc ccgcagccgt
ctgatttgct ctcactgtcg 300tatacagagc tgccccgcca gtccgaggat
tgcttgtatg tcaatgtatt tgcgcctgac 360accccaagtc aaaatcttcc
tgtcatggtg tggattcacg gaggcgcttt ttatcttgga 420gcgggcagtg
agccattgta tgacggatca aaacttgcgg cacagggaga agtcattgtc
480gttacattga actatcggct ggggccgttt ggctttttgc acttgtcttc
gtttgatgag 540gcgtattccg ataaccttgg gcttttagac caagccgccg
cactgaaatg ggtgcgggag 600aatatttcag cgtttggcgg tgatcccgat
aacgtaacag tatttggaga atccgccggc 660gggatgagca ttgccgcgct
tctcgctatg cctgcggcaa aaggcctgtt ccagaaagca 720atcatggaaa
gcggcgcttc tcgaacgatg acgaaagaac aagcggcgag cacctcggca
780gcctttttac aggtccttgg gattaacgag ggccaattgg ataaattgca
tacggtttct 840gcagaagatt tgctaaaagc ggctgatcag cttcggattg
cagaaaaaga aaatatcttt 900cagctgttct tccagcccgc ccttgatcca
aaaacgctgc ctgctgaacc agaaaaagcg 960atctcagaag gggctgcttc
cggcattcct ctattgattg gaacaacccg tgatgaagga 1020tatttatttt
tcaccccgga ttcagacgtt cattctcagg aaacgcttga tgcagcactc
1080gagtatttac tagggaagcc gctggcagag aaagctgccg atttgtatcc
gcgttctctg 1140gaaagccaaa ttcatatgat gactgattta ttattttggc
gccctgccgt cgcctatgca 1200tccgcacagt ctcattacgc ccctgtctgg
atgtacaggt tcgattggca cccgaagaag 1260ccgccgtaca ataaagcgtt
tcacgcatta gagcttcctt ttgtctttgg aaatctggac 1320ggattggaac
gaatggcaaa agcggagatt acggatgagg tgaaacagct ttctcacacg
1380atacaatcag cgtggatcac gttcgctaaa acaggaaacc caagcaccga
agctgtgaat 1440tggccgacgt atcatgaaga aacgagagag acgctgattt
tagattcaga gattacgatc 1500gaaaacgatc ccgaatctga aaaaaggcag
aagctattcc cttcaaaagg agaataa 155781557DNABacillus licheniformis
8atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg
60atggcattca gcgattccgc ttctgctatg tctcataaaa cagtaacaac tcaatacggc
120aaagtaaaag gcacaacaga aaacggcgta cataaatgga aaggcatccc
ctatgccaaa 180cctcctgtcg ggccattgcg ttttaaagca ccggaacctc
ctgaagcgtg ggagaacgaa 240ctggacgcaa cagcatacgg ctctatttgc
ccgcagccgt ctgatttgct gtcactttcg 300tatactgagc tgccccgcca
gtctgaggat tgcttgtata tcaatgtatt tgcgcctgat 360actccaagtc
aaaacctgcc tgtcatggta tggattcacg gcggcgcttt ttatcttgga
420gcgggcagtg agccattata tgatgggtca agacttgcgg cgcagggaga
agtcattgtc 480gttacactga attatcgtct ggggccgttt ggatttttac
atttgtcttc gtttgaagag 540acgtattccg ataaccttgg gcttttggac
caagccgccg cactgaaatg ggtgcgagac 600aatatctcag catttggcgg
tgatccggat aacgtaacag tatttggaga atcagcaggc 660ggcatgagca
ttgccgcgct gctcgcaatg cctgcggcaa aaggcctgtt ccagaaagca
720atcatggaaa gtggcgcttc cagaacgatg acaaaagaaa aagcggctag
caccgcggca 780gcctttttag aggtccttgg gattgacgag agccaattgg
acaggttgca tactgtatct 840gcggaagatt tgcttaaagc ggccgatcag
cttcggaaag cagaaaatga aaatctcttt 900cagctgttct tccagcccgc
ccttgatccg aaaacgctgc ctgctgaacc agaaaaagcg 960atcgcagagg
gtgctgctgc cggcattccg ctgttaatcg gaacaaaccg cgatgaagga
1020tatttatttt tcaccccgga ctcagacgtt cattctcagg aaacgtttga
tgccgcgctt 1080gtgtatttat tagggcagcc gctggcagag aaagccgccg
atctgtatcc gcgttcgctg 1140gaaagccaaa ttcatatgat gactgatttg
ttattttggc gcccggccgt cgcctgtgcc 1200tccgcacagt cccattacgc
gcctgtctgg atgtaccgat tcgattggca ctctgataag 1260ccgccgtata
ataaagcgtt tcacgcatta gagcttcctt ttgttttcgg aaatctggac
1320gggttagaac ggatggcaaa agcagagatt acggatgaag tgaaacagct
ctctcacacc 1380atacaatcag catggatcac gttcgccaaa acagggaacc
caagcactga agatgtaaaa 1440tggccggcgt atcatgagga aacaagagag
acgctgattt taaattcaga gattgcgatt 1500gaaaacgacc ctgaagctga
aaaaaggcag aaactattcc cttcacaagg agaataa 1557929PRTArtificialSIGNAL
9Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met1 5
10 15Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala 20
251087DNAArtificialsig_peptide 10atgatgagga aaaagagttt ttggcttggg
atgctgacgg ccttcatgct cgtgttcacg 60atggcattca gcgattccgc ttctgct
8711489PRTBacillus subtilis 11Met Thr His Gln Ile Val Thr Thr Gln
Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp
Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe
Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala
Thr Ala Tyr Gly Ser Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser
Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu
Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Lys Asn 85 90 95Leu Pro
Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105
110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu
115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly
Phe Leu 130 135 140His Leu Ser Ser Phe Asn Glu Ala Tyr Ser Asp Asn
Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val
Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val
Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala
Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala
Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln
Ala Ala Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230
235 240Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu
Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn
Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr
Leu Pro Glu Glu Pro 275 280 285Glu Lys Ala Ile Ala Glu Gly Ala Ala
Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly
Tyr Leu Phe Phe Thr Pro Asp Ser Asp305 310 315 320Val His Ser Gln
Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro
Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345
350Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val
355 360 365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met
Tyr Arg 370 375 380Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys
Ala Phe His Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn
Leu Asp Gly Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp
Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Ile
Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn
Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu
Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470
475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu 48512490PRTBacillus
licheniformis 12Met Tyr Asp Thr Thr Val Glu Thr Arg Phe Gly Lys Leu
Lys Gly Arg1 5 10 15Ala Glu Asn Gly Val Arg Ile Phe Lys Gly Val Pro
Tyr Ala Lys Pro 20 25 30Pro Val Gly Asp Leu Arg Phe Arg Glu Pro Gln
Arg Met Glu Ala Trp 35 40 45Glu Gly Glu Leu Asp Ala Phe Gln Phe Gly
Pro Val Cys Pro Gln Pro 50 55 60Asp Gly Val Leu Pro Glu Ser Ala Gly
Val Gln Lys Ser Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Val Tyr Ala
Pro Glu Glu Ala Asp Gly Asp Leu Pro 85 90 95Val Met Val Trp Ile His
Gly Gly Ala Phe Tyr Arg Gly Ala Gly Ser 100 105 110Glu Pro Leu Tyr
Asp Gly Thr Gln Leu Ala Lys Gln Gly Lys Val Ile 115 120 125Val Val
Thr Ile Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu His Leu 130 135
140Ser Ser Ile Asp Asp Ser Tyr Ser Ser Asn Leu Gly Leu Leu Asp
Gln145 150 155 160Ile Ala Ala Leu Glu Trp Val Lys Asp Asn Ile Ala
Phe Phe Gly Gly 165 170 175Asp Arg His His Ile Thr Val Phe Gly Glu
Ser Ala Gly Ser Met Ser 180 185 190Ile Ala Ser Leu Leu Ala Met Pro
Lys Ala Lys Gly Leu Phe Gln Gln 195 200 205Ala Ile Met Glu Ser Gly
Ala Ser Ala Thr Met Ser Asp Lys Leu Ala 210 215 220Lys Ala Ala Ala
Glu Arg Phe Leu Arg Ile Leu Asp Ile Asp His His225 230 235 240His
Leu Glu Arg Leu His Asp Val Ser Asp Gln Glu Leu Leu Glu Ala 245 250
255Ala Asp Gln Leu Arg Thr Leu Met Gly Glu Asn Ile Phe Glu Leu Ile
260 265 270Phe Leu Pro Ala Leu Asp Glu Lys Thr Leu Pro Leu Lys Pro
Glu Val 275 280 285Ala Val Ala Lys Gly Ala Ala Lys Glu Ile Asn Leu
Leu Ile Gly Thr 290 295 300Asn Arg Asp Glu Gly Val Leu Phe Phe Pro
Ser Asp Ser Asp Leu Leu305 310 315 320Pro Glu Ser Lys Ile Asn Glu
Ile Leu Glu Glu Tyr Met Gly Lys Glu 325 330 335Ala Ala Glu Ala Ala
Ser Ser Leu Tyr Pro Arg Ser Leu Glu Gly His 340 345 350Val Asp Met
Met Thr Asp Leu Ile Phe Trp His Pro Ser Val Val Phe 355 360 365Ala
Ser Ala Gln Ser Arg Tyr Ala Ser Val Phe Met Tyr Arg Phe Asp 370 375
380Trp His Ala Asp Ser Glu Gln Pro Pro Phe Asn Lys Ala Ala His
Gly385 390 395 400Leu Glu Ile Pro Phe Val Phe Gly Asn Met Asp Ile
Leu Glu Gln Leu 405 410 415Thr Gly Thr Lys Ala Gly Glu Glu Ala Gln
Leu Leu Ala Glu Gln Ile 420 425 430Gln Ala Ala Trp Val Ser Phe Ala
Arg Ser Gly Asn Pro Ser Thr Asp 435 440 445Asp Val Ser Trp Pro Asp
Tyr Asp Glu Asp Ser Arg Lys Thr Leu Ile 450 455 460Phe Asp Gln Glu
Val Ala Val Glu Ser Asp Pro Tyr Ser Asp Lys Arg465 470 475 480Lys
Met Leu Thr Ala Pro Asn Pro Gln Ile 485 49013489PRTBacillus
subtilis 13Met Thr His Gln Ile Val Thr Thr Gln Tyr Gly Lys Val Lys
Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp Lys Gly Ile Pro Tyr
Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe Lys Ala Pro Glu Pro
Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala Thr Ala Tyr Gly Pro
Val Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser Leu Ser Tyr Thr Glu
Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu Tyr Val Asn Val Phe
Ala Pro Asp Thr Pro Ser Gln Asn 85 90 95Leu Pro Val Met Val Trp Ile
His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105 110Gly Ser Glu Pro Leu
Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu 115 120 125Val Ile Val
Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Met 130 135 140His
Leu Ser Ser Phe Asp Glu Ala Tyr Ser Asp Asn Leu Gly Leu Leu145 150
155 160Asp Gln Ala Ala Ala Leu Lys Trp Val Arg Glu Asn Ile Ser Ala
Phe 165 170 175Gly Gly Asp Pro Asp Asn Val Thr Val Phe Gly Glu Ser
Ala Gly Gly 180 185 190Met Ser Ile Ala Ala Leu Leu Ala Met Pro Ala
Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala Ile Met Glu Ser Gly Ala
Ser Arg Thr Met Thr Lys Glu 210 215 220Gln Ala Ala Ser Thr Ala Ala
Ala Phe Leu Gln Val Leu Gly Ile Asn225 230 235 240Glu Ser Gln Leu
Asp Arg Leu His Thr Val Ala Ala Glu Asp Leu Leu 245 250 255Lys Ala
Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln 260 265
270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Glu Glu Pro
275 280 285Glu Lys Ser Ile Ala Glu Gly Ala Ala Ser Gly Ile Pro Leu
Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly Tyr Leu Phe Phe Thr
Ser Asp Ser Asp305 310 315 320Val Arg Ser Gln Glu Thr Leu Asp Ala
Ala Leu Glu Tyr Ser Leu Gly 325 330 335Lys Pro Leu Ala Glu Lys Ala
Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345 350Ser Gln Ile His Met
Val Thr Asp Leu Leu Phe Trp Arg Pro Ala Val 355 360 365Ala Phe Ala
Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg 370 375 380Phe
Asp Trp His Pro Glu Lys Pro Pro Tyr Asn Lys Ala Phe His Ala385 390
395 400Leu Glu Leu Pro Phe Val Phe Gly Asn Leu Asp Gly Leu Glu Arg
Met 405 410 415Ala Lys Ala Glu Ile Thr Asp Glu Val Lys Gln Leu Ser
His Thr Ile 420 425 430Gln Ser Ala Trp Ile Thr Phe Ala Lys Thr Gly
Asn Pro Ser Thr Glu 435 440 445Ala Val Asn Trp Pro Ala Tyr His Glu
Glu Thr Arg Glu Thr Val Ile 450 455 460Leu Asp Ser Glu Ile Thr Ile
Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470 475 480Gln Lys Leu Phe
Pro Ser Lys Gly Glu 48514489PRTBacillus subtilis 14Met Thr His Gln
Ile Val Thr Thr Gln Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn
Gly Val His Lys Trp Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val
Gly Gln Trp Arg Phe Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu
Asp Val Leu Asp Ala Thr Ala Tyr Gly Pro Ile Cys Pro Gln Pro 50 55
60Ser Asp Leu Leu Ser Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65
70 75 80Asp Cys Leu Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Gln
Asn 85 90 95Leu Pro Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu
Gly Ala 100 105 110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala
Ala Gln Gly Glu 115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu
Gly Pro Phe Gly Phe Leu 130 135 140His Leu Ser Ser Phe Asp Glu Ala
Tyr Ser Asp Asn Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala
Leu Lys Trp Val Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp
Pro Asp Asn Val Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met
Ser Ile Ala Ala Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200
205Gln Lys Ala Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu
210 215 220Gln Ala Ala Ser Thr Ala Ala Ala Phe Leu Gln Val Leu Gly
Ile Asn225 230 235 240Glu Ser Gln Leu Asp Arg Leu His Thr Val Ala
Ala Glu Asp Leu Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala
Glu Lys Glu Asn Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu
Asp Pro Lys Thr Leu Pro Glu Glu Pro 275 280 285Glu Lys Ser Ile Ala
Glu Gly Ala Ala Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr
Arg Asp Glu Gly Tyr Leu Phe Phe Thr Pro Asp Ser Asp305 310 315
320Val His Ser Gln Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly
325 330 335Lys Pro Leu Ala Glu Lys Ala Ala Asp Leu Tyr Pro Arg Ser
Leu Glu 340 345 350Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp
Arg Pro
Ala Val 355 360 365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val
Trp Met Tyr Arg 370 375 380Phe Asp Trp His Pro Glu Lys Pro Pro Tyr
Asn Lys Ala Phe His Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe
Gly Asn Leu Asp Gly Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile
Thr Asp Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala
Trp Ile Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala
Val Asn Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Val Ile 450 455
460Leu Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys
Arg465 470 475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu
48515489PRTBacillus subtilis 15Met Thr His Gln Ile Val Thr Thr Gln
Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp
Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe
Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala
Thr Ala Tyr Gly Ser Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser
Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu
Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Lys Asn 85 90 95Leu Pro
Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105
110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu
115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly
Phe Leu 130 135 140His Leu Ser Ser Phe Asn Glu Ala Tyr Ser Asp Asn
Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val
Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val
Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala
Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala
Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln
Ala Ala Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230
235 240Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu
Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn
Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr
Leu Pro Glu Glu Pro 275 280 285Glu Lys Ala Ile Ala Glu Gly Ala Ala
Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly
Tyr Leu Phe Phe Thr Pro Asp Ser Asp305 310 315 320Val His Ser Gln
Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro
Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345
350Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val
355 360 365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met
Tyr Arg 370 375 380Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys
Ala Phe His Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn
Leu Asp Gly Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp
Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Ile
Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn
Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu
Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470
475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu 48516489PRTBacillus
subtilis 16Met Thr His Gln Ile Val Thr Thr Gln Tyr Gly Lys Val Lys
Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp Lys Gly Ile Pro Tyr
Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe Lys Ala Pro Glu Pro
Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala Thr Ala Tyr Gly Ser
Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser Leu Ser Tyr Thr Glu
Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu Tyr Val Asn Val Phe
Ala Pro Asp Thr Pro Ser Lys Asn 85 90 95Leu Pro Val Met Val Trp Ile
His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105 110Gly Ser Glu Pro Leu
Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu 115 120 125Val Ile Val
Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu 130 135 140His
Leu Ser Ser Phe Asn Glu Ala Tyr Ser Asp Asn Leu Gly Leu Leu145 150
155 160Asp Gln Ala Ala Ala Leu Lys Trp Val Arg Glu Asn Ile Ser Ala
Phe 165 170 175Gly Gly Asp Pro Asp Asn Val Thr Val Phe Gly Glu Ser
Ala Gly Gly 180 185 190Met Ser Ile Ala Ala Leu Leu Ala Met Pro Ala
Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala Ile Met Glu Ser Gly Ala
Ser Arg Thr Met Thr Lys Glu 210 215 220Gln Ala Ala Ser Thr Ser Ala
Ala Phe Leu Gln Val Leu Gly Ile Asn225 230 235 240Glu Gly Gln Leu
Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu Leu 245 250 255Lys Ala
Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn Ile Phe Gln 260 265
270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr Leu Pro Glu Glu Pro
275 280 285Glu Lys Ala Ile Ala Glu Gly Ala Ala Ser Gly Ile Pro Leu
Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly Tyr Leu Phe Phe Thr
Pro Asp Ser Asp305 310 315 320Val His Ser Gln Glu Thr Leu Asp Ala
Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro Leu Ala Glu Lys Val
Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345 350Ser Gln Ile His Met
Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val 355 360 365Ala Tyr Ala
Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met Tyr Arg 370 375 380Phe
Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys Ala Phe His Ala385 390
395 400Leu Glu Leu Pro Phe Val Phe Gly Asn Leu Asp Gly Leu Glu Arg
Met 405 410 415Ala Lys Ala Glu Ile Thr Asp Glu Val Lys Gln Leu Ser
His Thr Ile 420 425 430Gln Ser Ala Trp Ile Thr Phe Ala Lys Thr Gly
Asn Pro Ser Thr Glu 435 440 445Ala Val Asn Trp Pro Ala Tyr His Glu
Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu Asp Ser Glu Ile Thr Ile
Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470 475 480Gln Lys Leu Phe
Pro Ser Lys Gly Glu 48517489PRTBacillus subtilis 17Met Thr His Gln
Ile Val Thr Thr Gln Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn
Gly Val His Lys Trp Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val
Gly Gln Trp Arg Phe Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu
Asp Val Leu Asp Ala Thr Ala Tyr Gly Ser Ile Cys Pro Gln Pro 50 55
60Ser Asp Leu Leu Ser Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65
70 75 80Asp Cys Leu Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Lys
Asn 85 90 95Leu Pro Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu
Gly Ala 100 105 110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala
Ala Gln Gly Glu 115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu
Gly Pro Phe Gly Phe Leu 130 135 140His Leu Ser Ser Phe Asn Glu Ala
Tyr Ser Asp Asn Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala
Leu Lys Trp Val Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp
Pro Asp Asn Val Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met
Ser Ile Ala Ala Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200
205Gln Lys Ala Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu
210 215 220Gln Ala Ala Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly
Ile Asn225 230 235 240Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser
Ala Glu Asp Leu Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala
Glu Lys Glu Asn Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu
Asp Pro Lys Thr Leu Pro Glu Glu Pro 275 280 285Glu Lys Ala Ile Ala
Glu Gly Ala Ala Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr
Arg Asp Glu Gly Tyr Leu Phe Phe Thr Pro Asp Ser Asp305 310 315
320Val His Ser Gln Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly
325 330 335Lys Pro Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser
Leu Glu 340 345 350Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp
Arg Pro Ala Val 355 360 365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala
Pro Val Trp Met Tyr Arg 370 375 380Phe Asp Trp His Pro Lys Lys Pro
Pro Tyr Asn Lys Ala Phe His Ala385 390 395 400Leu Glu Leu Pro Phe
Val Phe Gly Asn Leu Asp Gly Leu Glu Arg Met 405 410 415Ala Lys Ala
Glu Ile Thr Asp Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln
Ser Ala Trp Ile Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440
445Ala Val Asn Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Leu Ile
450 455 460Leu Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu
Lys Arg465 470 475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu
48518489PRTBacillus subtilis 18Met Thr His Gln Ile Val Thr Thr Gln
Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys Trp
Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg Phe
Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp Ala
Thr Ala Tyr Gly Ser Ile Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu Ser
Leu Ser Tyr Thr Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys Leu
Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Lys Asn 85 90 95Leu Pro
Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105
110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu
115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly
Phe Leu 130 135 140His Leu Ser Ser Phe Asn Glu Ala Tyr Ser Asp Asn
Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val
Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val
Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala
Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala
Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln
Ala Ala Ser Thr Ser Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230
235 240Glu Gly Gln Leu Asp Lys Leu His Thr Val Ser Ala Glu Asp Leu
Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn
Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr
Leu Pro Glu Glu Pro 275 280 285Glu Lys Ala Ile Ala Glu Gly Ala Ala
Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly
Tyr Leu Phe Phe Thr Pro Asp Ser Asp305 310 315 320Val His Ser Gln
Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro
Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345
350Ser Gln Ile His Met Met Thr Asp Leu Leu Phe Trp Arg Pro Ala Val
355 360 365Ala Tyr Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met
Tyr Arg 370 375 380Phe Asp Trp His Pro Lys Lys Pro Pro Tyr Asn Lys
Ala Phe His Ala385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn
Leu Asp Gly Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp
Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Ile
Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn
Trp Pro Ala Tyr His Glu Glu Thr Arg Glu Thr Leu Ile 450 455 460Leu
Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470
475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu 48519490PRTBacillus
licheniformis 19Met Tyr Asp Thr Thr Val Glu Thr Arg Phe Gly Lys Leu
Lys Gly Arg1 5 10 15Ala Glu Asn Gly Val Arg Ile Phe Lys Gly Val Pro
Tyr Ala Lys Pro 20 25 30Pro Val Gly Asp Leu Arg Phe Arg Glu Pro Gln
Arg Met Glu Ala Trp 35 40 45Glu Gly Glu Leu Asp Ala Phe Gln Phe Gly
Pro Val Cys Pro Gln Pro 50 55 60Asp Gly Val Leu Pro Glu Ser Ala Gly
Val Gln Lys Ser Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Val Tyr Ala
Pro Glu Glu Ala Asp Gly Asp Leu Pro 85 90 95Val Met Val Trp Ile His
Gly Gly Ala Phe Tyr Arg Gly Ala Gly Ser 100 105 110Glu Pro Leu Tyr
Asp Gly Thr Gln Leu Ala Lys Gln Gly Lys Val Ile 115 120 125Val Val
Thr Ile Asn Tyr Arg Leu Gly Pro Phe Gly Phe Leu His Leu 130 135
140Ser Ser Ile Asp Asp Ser Tyr Ser Ser Asn Leu Gly Leu Leu Asp
Gln145 150 155 160Ile Ala Ala Leu Glu Trp Val Lys Asp Asn Ile Ala
Phe Phe Gly Gly 165 170 175Asp Arg His His Ile Thr Val Phe Gly Glu
Ser Ala Gly Ser Met Ser 180 185 190Ile Ala Ser Leu Leu Ala Met Pro
Lys Ala Lys Gly Leu Phe Gln Gln 195 200 205Ala Ile Met Glu Ser Gly
Ala Ser Ala Thr Met Ser Asp Lys Leu Ala 210 215 220Lys Ala Ala Ala
Glu Arg Phe Leu Arg Ile Leu Asp Ile Asp His His225 230 235 240His
Leu Glu Arg Leu His Asp Val Ser Asp Gln Glu Leu Leu Glu Ala 245 250
255Ala Asp Gln Leu Arg Thr Leu Met Gly Glu Asn Ile Phe Glu Leu Ile
260 265 270Phe Leu Pro Ala Leu Asp Glu Lys Thr Leu Pro Leu Lys Pro
Glu Val 275 280 285Ala Val Ala Lys Gly Ala Ala Lys Glu Ile Asn Leu
Leu Ile Gly Thr 290 295 300Asn Arg Asp Glu Gly Val Leu Phe Phe Pro
Ser Asp Ser Asp Leu Leu305 310 315 320Pro Glu Ser Lys Ile Asn Glu
Ile Leu Glu Glu Tyr Met Gly Lys Glu 325 330 335Ala Ala Glu Ala Ala
Ser Ser Leu Tyr Pro Arg Ser Leu Glu Gly His 340 345 350Val Asp Met
Met Thr Asp Leu Ile Phe Trp His Pro Ser Val Val Phe 355 360 365Ala
Ser Ala Gln
Ser Arg Tyr Ala Ser Val Phe Met Tyr Arg Phe Asp 370 375 380Trp His
Ala Asp Ser Glu Gln Pro Pro Phe Asn Lys Ala Ala His Gly385 390 395
400Leu Glu Ile Pro Phe Val Phe Gly Asn Met Asp Ile Leu Glu Gln Leu
405 410 415Thr Gly Thr Lys Ala Gly Glu Glu Ala Gln Leu Leu Ala Glu
Gln Ile 420 425 430Gln Ala Ala Trp Val Ser Phe Ala Arg Ser Gly Asn
Pro Ser Thr Asp 435 440 445Asp Val Ser Trp Pro Asp Tyr Asp Glu Asp
Ser Arg Lys Thr Leu Ile 450 455 460Phe Asp Gln Glu Val Ala Val Glu
Ser Asp Pro Tyr Ser Asp Lys Arg465 470 475 480Lys Met Leu Thr Ala
Pro Asn Pro Gln Ile 485 49020455PRTStaphylococcus epidermidis 20Met
Cys Ser Asn Met Val Gln Val Lys Ile Gly Asn Cys Thr Ile Asn1 5 10
15Gly Leu His Lys Lys Asn Ile Asp Val Phe Leu Gly Ile Pro Tyr Ala
20 25 30Lys Ser Phe Asn Lys Ile Ser Arg Phe Gln His Ser Lys Leu Met
Glu 35 40 45Leu Ser Lys Pro Met Ile Asp Ala Thr His Ile Gln Ser Ile
Pro Pro 50 55 60Gln Pro Tyr Asn Ser Leu Glu Asp Phe Phe Ser Met Thr
Asp Ser Ser65 70 75 80Phe Asn Ser Phe Lys Gln Asn Asp Tyr Cys Leu
Phe Leu Asn Ile Trp 85 90 95Lys Pro Ser Ser Asn Gln Asn His Leu Pro
Val Val Ile Tyr Phe Tyr 100 105 110Gly Gly Ser Phe Leu Gln Gly His
Gly Thr Ala Glu Leu Tyr Cys Pro 115 120 125Glu His Ile Val Glu Gln
Glu Asn Ile Ile Val Val Thr Phe Asn Tyr 130 135 140Arg Leu Gly Ala
Leu Gly Tyr Leu Asp Trp Ser Tyr Phe Asn Gln His145 150 155 160Leu
Asn Tyr Asn Asn Gly Ile Ser Asp Gln Ile Asn Val Leu Arg Trp 165 170
175Val His Gln Tyr Ile Glu His Phe Gly Gly Asp Ser Asn Asn Val Thr
180 185 190Leu Met Gly Gln Ser Ala Gly Ser Met Ser Ile Met Thr Leu
Met Gln 195 200 205Met Pro Glu Leu Asp Asp Tyr Tyr His Lys Val Met
Leu Leu Ser Gly 210 215 220Thr Leu Thr Thr Asp Thr Pro Leu Asn Ala
His Thr Lys Val Gln His225 230 235 240Phe Ser Gln Leu Met Arg His
Tyr Phe Pro Asn Lys Thr Leu Lys Thr 245 250 255Leu Thr Ser Asp Asp
Ile Leu Tyr Leu Met Glu Ser Gln Lys Ile Glu 260 265 270Arg Gly Arg
Ser Arg Gly Leu Asp Leu Ile Tyr Gln Pro Ile Lys Asp 275 280 285His
His Met Ser Arg Ser Ile Lys Lys Phe Pro Lys Pro Thr Phe Met 290 295
300Ser Tyr Thr His Asp Glu Gly Asp Ile Tyr Ile Glu Asp Ala Thr
Arg305 310 315 320Thr Leu Pro Ser Glu Arg Phe Ile His Leu Met Ser
Gln Tyr Gly Thr 325 330 335His Val Glu Lys Asn Asp Ala Leu Thr Met
Lys Gln Gln Arg Asn Leu 340 345 350Ile Thr Glu Tyr Cys Phe Val Arg
Pro Ile Tyr Leu Phe Leu Asn Lys 355 360 365Met Asn Ser Cys Asp Thr
Trp Leu Ala Arg Phe Asp Trp His Gln Pro 370 375 380His Thr Ser Tyr
Phe Lys Ser Ala Tyr His Ile Leu Asp Leu Val Phe385 390 395 400Trp
Phe Gly His Leu Ser Ile Leu Thr Lys Asn His Tyr Ser Ile Thr 405 410
415Gln His Asp Met Asn Leu Ser Arg Asn Met Ile Ser Asp Leu Ala Tyr
420 425 430Phe Ala Arg Lys Gly Lys Met Pro Trp Lys Cys Tyr Glu Pro
Gln His 435 440 445Gln Ala Leu His Ile Tyr Arg 450
45521450PRTStaphylococcus aureus 21Met Lys Ile Asn Thr Thr Gly Gly
Gln Ile His Gly Ile Thr Gln Asp1 5 10 15Gly Leu Asp Ile Phe Leu Gly
Ile Pro Tyr Ala Glu Pro Pro Val His 20 25 30Asp Asn Arg Phe Lys His
Ser Thr Leu Lys Thr Gln Trp Ser Glu Pro 35 40 45Ile Asp Ala Thr Glu
Ile Gln Pro Ile Pro Pro Gln Pro Asp Asn Lys 50 55 60Leu Glu Asp Phe
Phe Ser Ser Gln Ser Thr Thr Phe Thr Glu His Glu65 70 75 80Asp Cys
Leu Tyr Leu Asn Ile Trp Lys Gln His Asn Asp Gln Thr Lys 85 90 95Lys
Pro Val Ile Ile Tyr Phe Tyr Gly Gly Ser Phe Glu Asn Gly His 100 105
110Gly Lys Ala Glu Leu Tyr Gln Pro Ala His Leu Val Gln Asn Asn Asp
115 120 125Ile Ile Val Ile Thr Cys Asn Tyr Arg Leu Gly Ala Leu Gly
Tyr Leu 130 135 140Asp Trp Ser Tyr Phe Asn Lys Asp Phe His Ser Asn
Asn Gly Leu Ser145 150 155 160Asp Gln Ile Asn Val Ile Lys Trp Val
His Gln Phe Ile Glu Ser Phe 165 170 175Gly Gly Asp Ala Asn Asn Ile
Thr Leu Met Gly Gln Ser Ala Gly Ser 180 185 190Met Ser Ile Leu Thr
Leu Leu Lys Ile Pro Asp Ile Glu Pro Tyr Phe 195 200 205His Lys Val
Val Leu Leu Ser Gly Ala Leu Arg Leu Asp Thr Leu Glu 210 215 220Ser
Ala Arg Asn Lys Ala Gln His Phe Gln Lys Met Met Leu Asp Tyr225 230
235 240Leu Asp Thr Asp Asp Val Thr Ser Leu Ser Thr Asn Asp Ile Leu
Met 245 250 255Leu Met Ala Lys Leu Lys Gln Ser Arg Gly Pro Ser Lys
Gly Leu Asp 260 265 270Leu Ile Tyr Ala Pro Ile Lys Thr Asp Tyr Ile
Gln Asn Asn Tyr Pro 275 280 285Thr Thr Lys Pro Ile Phe Ala Cys Tyr
Thr Lys Asp Glu Gly Asp Ile 290 295 300Tyr Ile Thr Ser Glu Gln Lys
Lys Leu Ser Pro Gln Arg Phe Ile Asp305 310 315 320Ile Met Glu Leu
Asn Asp Ile Pro Leu Lys Tyr Glu Asp Val Gln Thr 325 330 335Ala Lys
Gln Gln Ser Leu Ala Ile Thr His Cys Tyr Phe Lys Gln Pro 340 345
350Met Lys Gln Phe Leu Gln Gln Leu Asn Ile Gln Asp Ser Asn Ala Gln
355 360 365Leu Trp Leu Ala Glu Phe Ala Trp His Asp Thr Ser Ser Ala
His Tyr 370 375 380Arg Ser Ala Tyr His Ile Leu Asp Met Val Phe Trp
Phe Gly Asn Leu385 390 395 400Gln Ile Leu Ala Ala His Gln Tyr Pro
Thr Thr Ala His Leu Lys Phe 405 410 415Leu Ser Arg Gln Met Gln Asn
Asp Leu Ala Asn Phe Ala Lys Ser Gly 420 425 430Lys Met Pro Trp Pro
Met Tyr His Asn Glu Arg Arg Tyr Tyr Arg Thr 435 440 445Tyr Gln
45022492PRTStreptomyces avermitilis 22Met Arg Gly Arg Leu Glu Gly
Gly Leu Ala Val Phe Arg Gly Val Pro1 5 10 15Phe Ala Glu Pro Pro Val
Gly Asp Ala Arg Phe Ala Ala Pro Arg Pro 20 25 30Val Arg Ala Trp Asp
Gly Thr Arg Asp Ala Phe Ala Phe Gly Pro Pro 35 40 45Pro Pro Gln Glu
Thr Gly Ile Gln Gly Arg Ala Ala Leu Leu Asp Ala 50 55 60Pro Thr Gly
Asp Asp Trp Leu Thr Val Asn Val Trp Thr Pro Asp Pro65 70 75 80Asp
Pro Gly Ala Arg Arg Pro Val Met Val Trp Ile Tyr Gly Gly Ala 85 90
95Tyr Lys Leu Gly His Ser Gly Ser Pro Gly Tyr Asp Ala Arg Arg Ile
100 105 110Ala Arg Asp Gly Asp Val Val Val Val Thr Leu Asn Tyr Arg
Val Gly 115 120 125Ile Glu Gly Phe Ala Arg Val Asp Gly Ala Pro Ala
Asn Arg Gly Leu 130 135 140Leu Asp Gln Val Ala Ala Leu Glu Trp Val
Arg Glu Asn Ile Thr Ala145 150 155 160Phe Gly Gly Asp Pro Gly Arg
Val Thr Val Phe Gly Glu Ser Ala Gly 165 170 175Ala Gly Ser Ile Ala
Ser Leu Leu Ala Met Pro Ser Ala Ser Gly Leu 180 185 190Phe Arg Arg
Ala Ile Ala Gln Ser Val Pro Gly Thr Tyr Phe Ser Asp 195 200 205Glu
Leu Ala Lys Asp Ile Ala Ala Ala Ile Ala Ala Glu Ala Gly Leu 210 215
220Arg Pro Thr Ala Ala Asp Leu Ser Thr Val Asp Pro Arg Gln Leu
Pro225 230 235 240Ala Ala Gly Glu Ala Leu Ala Ala Thr Met Arg Gln
Tyr Glu Asp Arg 245 250 255Trp Gly Pro Val Val His Thr Leu Thr Pro
Phe Ser Pro Val Val Asp 260 265 270Gly Glu Val Leu Pro Thr Thr Pro
Trp Gln Ala Leu Ala Ala Gly Thr 275 280 285Ala Arg Asp Val Glu Leu
Ile Val Gly His Asn Ser Glu Glu Phe Arg 290 295 300Leu Phe Val Leu
Leu Ser Gly Gln Leu Gly Lys Ile Thr Asp Gly Glu305 310 315 320Ala
Arg Ala Ala Leu Arg Arg Phe Gly Pro Gly Pro Asp Ala Glu Gln 325 330
335Ala Tyr Arg Thr Gly Phe Pro Asp Ala Ser Pro Gly Glu Leu Tyr Glu
340 345 350Arg Val Met Ser Asp Trp Leu Phe His Met Pro Ser Leu His
Leu Ala 355 360 365Glu Ala Gln Leu Thr Gly Gly Gly Arg Ala His Val
Tyr Glu Leu Thr 370 375 380Trp Pro Ala Pro Gly Asn Gly Gly Val Leu
Gly Ala Cys His Gly Leu385 390 395 400Asp Ile Pro Leu Leu Phe Gly
Thr Phe Asp Ala Asp Leu Gly Ser Leu 405 410 415Leu Phe Ala Gly Thr
Glu Pro Ser Pro Glu Ala Glu Ala Leu Ser Ser 420 425 430Arg Phe Arg
Ala Ser Trp Thr Ala Phe Ala Arg Thr Gly Asp Pro Gly 435 440 445Trp
Pro Thr Tyr Asp Thr Glu Arg Arg Leu Val Gln Val Leu Asp Ala 450 455
460Ala Pro Glu Val Ile Pro Tyr Pro Glu Glu Thr Ser Arg Arg Leu
Trp465 470 475 480Glu Arg His Thr Phe Pro Ala Leu Pro Leu Ile Gln
485 49023515PRTCaulobacter crescentus 23Met Gly Phe Thr Ala Glu Ala
Arg Ser Pro Val Val Ala Thr Thr Asn1 5 10 15Gly Lys Val Arg Gly Tyr
Leu Asp Gly Glu Val Ser Val Phe Lys Gly 20 25 30Leu Arg Tyr Gly Ala
Asp Thr Gly Gly Ala Arg Arg Phe Met Pro Pro 35 40 45Val Lys Pro Glu
Pro Trp Thr Glu Val Lys Asp Ala Leu Ala Tyr Gly 50 55 60Pro Ala Ser
Met Gln Thr Gly Lys Gly Glu Glu Gly Glu Thr Leu Ser65 70 75 80Glu
Asp Cys Leu Phe Leu Asn Val Trp Thr Pro Ala Arg Ala Ser Arg 85 90
95Lys Thr Gly Leu Ala Asp Gly Ala Lys Arg Pro Val Met Phe Tyr Ile
100 105 110His Gly Gly Ala Tyr Asn Gly Gly Ser Gly Ala Ser Pro Trp
Tyr Glu 115 120 125Gly Thr Lys Leu Ala Lys Arg Gly Asp Val Val Val
Val Thr Val Asn 130 135 140His Arg Leu Asn Ala Phe Gly Tyr Leu Tyr
Leu Ala Arg Leu Phe Asn145 150 155 160Ala Pro Ser Val Ala Asp Ser
Gly Asn Val Gly Gln Leu Asp Leu Val 165 170 175Leu Ala Leu Gln Trp
Val Arg Asp Asn Ile Ala Arg Phe Gly Gly Asp 180 185 190Pro Asp Cys
Val Met Leu Phe Gly Gln Ser Gly Gly Gly Ala Lys Ile 195 200 205Ala
Thr Leu Met Ala Met Pro Ser Ala Lys Gly Leu Phe His Arg Ala 210 215
220Ala Thr Met Ser Gly Gln Gln Val Thr Val Gly Gly Pro Phe Asn
Ala225 230 235 240Thr Arg Arg Ala Lys Ala Phe Leu Asp Lys Leu Gly
Val Lys Asp Leu 245 250 255Ala Ala Leu Arg Ala Leu Pro Ala Ala Glu
Met Leu Ala Gly Leu Lys 260 265 270Ala Val Asp Pro Ile Ala Gly Ser
Gly Gly Val Tyr Val Gly Pro Val 275 280 285Leu Asp Gln Arg Ser Leu
Leu Arg His Pro Phe Phe Pro Asp Ala Ala 290 295 300Pro Gln Ser Leu
Ser Ile Pro Met Met Val Gly Asn Thr His Asp Glu305 310 315 320Thr
Lys Gly Phe Ile Gly Trp Asp Ala Lys Ala Phe Pro Gln Thr Trp 325 330
335Asp Glu Val Ile Ala Arg Leu Pro Gly Gln Phe Ala Ala Arg Ile Asp
340 345 350Ile Asp Pro Glu Thr Val Val Ala Phe Tyr Arg Gln Thr Tyr
Pro Asn 355 360 365Tyr Ser Pro Ala Asp Val Phe Phe Ala Ala Ser Thr
Ala Gly Arg Ser 370 375 380Trp Lys Ala Ala Ile Ile Gln Asp Glu Glu
Arg Ala Lys Ala Gly Ala385 390 395 400Pro Ala Phe Ala Tyr Gln Val
Asn Trp Arg Ser Pro Ile Gln Gly Gly 405 410 415Ile Phe Gly Ala Pro
His Thr Ile Asp Ile Gly Leu Val Phe Gly Thr 420 425 430Leu Asp Ala
Lys Gly Ser Ile Val Gly Thr Gly Pro Asp Ser Val Ala 435 440 445Met
Ser Asn Thr Met Ser Asp Ala Phe Ile Ala Phe Ala Arg Thr Gly 450 455
460Asp Pro Asn Gly Gly Ala Leu Pro Lys Trp Glu Pro Tyr Thr Leu
Pro465 470 475 480Arg Arg Gln Thr Met Val Phe Asp Thr Val Ser Arg
Leu Glu Asp Asp 485 490 495Pro Arg Gly Val Glu Arg Glu Phe Phe Asn
Arg Val Pro Phe Thr Gln 500 505 510Phe Gly Thr
51524497PRTClostridium acetobuylicum 24Met Gly Thr Ile Ala Glu Thr
Lys Tyr Gly Lys Leu Glu Gly Ile Lys1 5 10 15Glu Asn Gly Ile Asn Lys
Trp Leu Gly Ile Pro Tyr Ala Lys Pro Pro 20 25 30Val Gly Glu Leu Arg
Phe Lys Arg Thr Val Glu Cys Glu Pro Trp Asn 35 40 45Gly Val Arg Tyr
Ala Lys Lys His Gly Ser Lys Pro His Gln Phe Ala 50 55 60Asn Thr Ser
Glu Glu Val Gly Ile Glu Ser Glu Asp Cys Leu Tyr Met65 70 75 80Asn
Ile Trp Ala Pro Glu Asn Ala Lys Asn Ser Pro Val Phe Val Trp 85 90
95Ile Tyr Gly Gly Ala Tyr Ala Met Gly Ser Cys Ser Glu Ala Tyr Tyr
100 105 110Asp Gly Thr Asn Phe Ala Lys Glu Gly Ile Val Tyr Val Ala
Phe Asn 115 120 125Tyr Arg Leu Gly Val Leu Gly Phe Tyr Asp Phe Thr
Met Tyr Asp Asp 130 135 140Ser Phe Asp Ser Asn Cys Gly Val Ser Asp
Gln Ile Met Ala Leu Lys145 150 155 160Trp Val Lys Glu Asn Ile Glu
Ala Phe Gly Gly Asp Pro Asn Asn Ile 165 170 175Thr Ile Ala Gly Glu
Ser Ala Gly Ala Ala Ser Val Thr Asn Met Leu 180 185 190Ala Val Pro
Lys Ala Lys Gly Leu Phe Asn Lys Ala Ile Ala Glu Ser 195 200 205Pro
Leu Pro Gly Cys Val Thr Ser His Asn Thr Ala Arg Leu Ile Thr 210 215
220Asp Ile Tyr Leu Lys Arg Leu Gly Leu Glu Ala Ser Glu Val His
Lys225 230 235 240Leu Lys Thr Met Glu Leu Glu Asp Ile Lys Lys Ala
Ala Leu Tyr Val 245 250 255Ile Asp Asp Thr Cys Ser Ser Tyr Pro Gly
Met Tyr Ile Pro Gly Pro 260 265 270Val Leu Asp Asp Leu Ile Pro Arg
Leu Pro Trp Glu Gly Ile Ala Leu 275 280 285Gly Ser Ser Lys Gly Val
Lys Leu Ile Ile Gly Thr Asn His Asp Glu 290 295 300Gly Thr Leu Phe
Ile Asn Lys Asn Lys Ser Met Leu Pro Gly Gly Trp305 310 315 320Lys
Asp Val Glu Arg Met Leu Arg Met Asn Lys Cys Phe Asp Ser Leu 325 330
335Pro Lys Ile His Lys Leu Tyr Asp Lys Phe Ser Glu Glu Met Ile Gln
340 345 350Ile Gln Glu Ile Met Lys Asp Arg Thr Phe Leu Val Asp Ser
Ile Lys 355 360 365Val Ala Asp Ala Gln Ser Glu Lys Asn Asp Thr Trp
Met Tyr Arg Phe 370 375 380Asp Tyr Ala Pro Ile Ser Ala Lys Leu Asn
Gly Leu Gly Ala Thr His385 390 395 400Ala Val Glu Val Ser Val Ala
Leu Asn Asn Thr Lys Gly Glu Gly Ile 405 410 415Ala Tyr Ser Phe
Trp
Arg Asp Thr Pro Glu Asp Ile Ile Lys Lys Phe 420 425 430Ile Glu Asn
Met His Met Ser Trp Val Asn Phe Ala Lys Thr Gly Asp 435 440 445Pro
Asn Gly Asn Leu Asp Ile Glu Trp Lys Lys Tyr Asp Ser Lys Ser 450 455
460Arg Thr Thr Phe Val Phe Asp Glu Glu Asn Lys Val Glu Asn Asn
Pro465 470 475 480Ala Lys Asp Ile Tyr Glu Thr Trp Arg Asp Ile Lys
Leu Tyr Thr Asp 485 490 495Ile25489PRTartificialprotein of unknown
origin, database entry gi 7546320 25Met Thr His Gln Ile Val Thr Thr
Gln Tyr Gly Lys Val Lys Gly Thr1 5 10 15Thr Glu Asn Gly Val His Lys
Trp Lys Gly Ile Pro Tyr Ala Lys Pro 20 25 30Pro Val Gly Gln Trp Arg
Phe Lys Ala Pro Glu Pro Pro Glu Val Trp 35 40 45Glu Asp Val Leu Asp
Ala Thr Val Tyr Gly Pro Val Cys Pro Gln Pro 50 55 60Ser Asp Leu Leu
Ser Leu Ser Tyr Lys Glu Leu Pro Arg Gln Ser Glu65 70 75 80Asp Cys
Leu Tyr Val Asn Val Phe Ala Pro Asp Thr Pro Ser Gln Asn 85 90 95Leu
Pro Val Met Val Trp Ile His Gly Gly Ala Phe Tyr Leu Gly Ala 100 105
110Gly Ser Glu Pro Leu Tyr Asp Gly Ser Lys Leu Ala Ala Gln Gly Glu
115 120 125Val Ile Val Val Thr Leu Asn Tyr Arg Leu Gly Pro Phe Gly
Phe Met 130 135 140His Leu Ser Ser Phe Asp Glu Ala Tyr Ser Asp Asn
Leu Gly Leu Leu145 150 155 160Asp Gln Ala Ala Ala Leu Lys Trp Val
Arg Glu Asn Ile Ser Ala Phe 165 170 175Gly Gly Asp Pro Asp Asn Val
Thr Val Phe Gly Glu Ser Ala Gly Gly 180 185 190Met Ser Ile Ala Ala
Leu Leu Ala Met Pro Ala Ala Lys Gly Leu Phe 195 200 205Gln Lys Ala
Ile Met Glu Ser Gly Ala Ser Arg Thr Met Thr Lys Glu 210 215 220Gln
Ala Ala Ser Thr Ala Ala Ala Phe Leu Gln Val Leu Gly Ile Asn225 230
235 240Glu Ser Gln Leu Asp Arg Leu His Thr Val Ala Ala Glu Asp Leu
Leu 245 250 255Lys Ala Ala Asp Gln Leu Arg Ile Ala Glu Lys Glu Asn
Ile Phe Gln 260 265 270Leu Phe Phe Gln Pro Ala Leu Asp Pro Lys Thr
Leu Pro Glu Glu Pro 275 280 285Glu Lys Ser Ile Ala Glu Gly Ala Ala
Ser Gly Ile Pro Leu Leu Ile 290 295 300Gly Thr Thr Arg Asp Glu Gly
Tyr Phe Phe Phe Thr Pro Asp Ser Asp305 310 315 320Val Tyr Ser Gln
Glu Thr Leu Asp Ala Ala Leu Glu Tyr Leu Leu Gly 325 330 335Lys Pro
Leu Ala Glu Lys Val Ala Asp Leu Tyr Pro Arg Ser Leu Glu 340 345
350Ser Gln Ile His Met Val Thr Asp Leu Leu Phe Trp Arg Pro Ala Val
355 360 365Ala Phe Ala Ser Ala Gln Ser His Tyr Ala Pro Val Trp Met
Tyr Arg 370 375 380Phe Asp Trp His Pro Glu Lys Pro Pro Tyr Asn Lys
Ala Phe His Thr385 390 395 400Leu Glu Leu Pro Phe Val Phe Gly Asn
Leu Asp Glu Leu Glu Arg Met 405 410 415Ala Lys Ala Glu Ile Thr Asp
Glu Val Lys Gln Leu Ser His Thr Ile 420 425 430Gln Ser Ala Trp Thr
Thr Phe Ala Lys Thr Gly Asn Pro Ser Thr Glu 435 440 445Ala Val Asn
Trp Pro Ala Tyr His Glu Glu Ser Arg Glu Thr Val Ile 450 455 460Leu
Asp Ser Glu Ile Thr Ile Glu Asn Asp Pro Glu Ser Glu Lys Arg465 470
475 480Gln Lys Leu Phe Pro Ser Lys Gly Glu 48526320PRTBurkholderia
cepacia 26Ala Asp Asn Tyr Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val
His Gly1 5 10 15Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu Tyr
Trp Tyr Gly 20 25 30Ile Gln Glu Asp Leu Gln Gln Arg Gly Ala Thr Val
Tyr Val Ala Asn 35 40 45Leu Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn
Gly Arg Gly Glu Gln 50 55 60Leu Leu Ala Tyr Val Lys Thr Val Leu Ala
Ala Thr Gly Ala Thr Lys65 70 75 80Val Asn Leu Val Gly His Ser Gln
Gly Gly Leu Thr Ser Arg Tyr Val 85 90 95Ala Ala Val Ala Pro Asp Leu
Val Ala Ser Val Thr Thr Ile Gly Thr 100 105 110Pro His Arg Gly Ser
Glu Phe Ala Asp Phe Val Gln Gly Val Leu Ala 115 120 125Tyr Asp Pro
Thr Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn 130 135 140Val
Phe Gly Ile Leu Thr Ser Ser Ser Asn Asn Thr Asn Gln Asp Ala145 150
155 160Leu Ala Ala Leu Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr
Asn 165 170 175Gln Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser
Cys Gln Thr 180 185 190Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr
His Leu Leu Tyr Ser 195 200 205Trp Ala Gly Thr Ala Ile Gln Pro Thr
Ile Ser Val Phe Gly Val Thr 210 215 220Gly Ala Thr Asp Thr Ser Thr
Ile Pro Leu Val Asp Pro Ala Asn Ala225 230 235 240Leu Asp Pro Ser
Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Val 245 250 255Asn Arg
Gly Ser Gly Gln Asn Asp Gly Val Val Ser Lys Cys Ser Ala 260 265
270Leu Tyr Gly Gln Val Leu Ser Thr Ser Tyr Lys Trp Asn His Leu Asp
275 280 285Glu Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Asn Ala Glu
Asp Pro 290 295 300Val Ala Val Ile Arg Thr His Ala Asn Arg Leu Lys
Leu Ala Gly Val305 310 315 320271473DNABacillus licheniformis
27atgtatgata caactgtcga aacacgcttc ggaaagctga aaggcagagc ggaaaacgga
60gtccgtatct ttaaaggcgt tccatacgca aaacctcccg tcggcgacct aaggtttcgg
120gaaccgcagc gaatggaggc ctgggaaggt gagctggatg cttttcaatt
tggcccggtt 180tgtccgcagc ctgatggggt attgcctgag tcagcggggg
ttcaaaagtc tgaggattgc 240ctttatttaa atgtgtacgc acccgaagag
gcggacgggg atctgcctgt tatggtgtgg 300attcatgggg gcgcttttta
tcgcggcgcc ggaagtgaac cgctctatga cgggactcag 360cttgcaaagc
agggaaaggt gatcgtggtc accatcaatt atcgcctcgg tccgttcggt
420tttttgcatc tatcctcaat tgatgattcc tacagcagca atcttggcct
gctggatcaa 480atcgcggctc tcgagtgggt gaaagacaat atcgctttct
ttggcggaga ccgtcatcac 540attacggttt ttggagagtc ggcgggatcg
atgagcatcg cttcgctttt ggcgatgccg 600aaagcaaagg ggctttttca
acaggccatt atggaaagcg gggcttccgc aactatgtcc 660gataagcttg
cgaaagctgc agcagaaaga ttcttaagga ttctcgatat tgatcatcat
720catctggagc gccttcatga tgtatctgat caagaacttc ttgaagccgc
cgatcagctg 780cgcactttaa tgggagaaaa tatttttgaa ttgatttttc
tgcctgcgct tgacgaaaaa 840accttgccgc tgaagccgga ggtcgccgtc
gcaaaaggcg cggcaaaaga gatcaatcta 900ttaatcggaa caaaccgtga
tgaaggcgtc ttgttttttc cctctgattc ggatcttttg 960cctgagagca
agatcaacga gattttagaa gaatacatgg gtaaagaggc cgccgaagcc
1020gcctcctctc tgtatccgag gtcattggaa ggccatgttg atatgatgac
agatctgatc 1080ttttggcatc cgtctgttgt gttcgcttcg gctcaatcac
gatatgcatc tgtctttatg 1140taccggtttg actggcatgc ggattcagag
cagccgccgt tcaacaaagc tgcgcacggc 1200ttagagattc cgtttgtatt
tggaaatatg gacattttgg aacagctgac aggtacgaag 1260gccggtgaag
aagcgcagct gcttgctgaa cagatccagg ctgcctgggt gtcttttgcc
1320cgatccggaa atccgagcac cgatgatgtc agctggcctg attatgatga
agattcacgg 1380aaaacgctga tttttgatca agaggtcgca gttgaaagcg
atccttattc agataagaga 1440aagatgttga cagccccgaa cccgcagatt tag
1473
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